KR20110057166A - Antibody Against CCR2 - Google Patents

Abstract

The present invention provides an antibody comprising a human antibody and an antigen binding portion thereof which specifically bind to CCR2, in particular human CCR2, and which can function to inhibit CCR2. Anti-CCR2 antibodies include binding to the first and / or second extracellular loop of CCR2. The invention also provides human anti-CCR2 antibodies and antigen binding portions thereof. The present invention provides isolated heavy and light chain immunoglobulins derived from human anti-CCR2 antibodies and nucleic acid molecules encoding these immunoglobulins. The present invention provides a method of making a human anti-CCR2 antibody or antigen binding moiety, a composition comprising the antibody or antigen binding moiety, a method of using the antibody and an antigen binding moiety and a composition for diagnosis and treatment. The invention also provides methods of gene therapy utilizing nucleic acid molecules encoding heavy and / or light chain immunoglobulin molecules comprising human anti-CCR2 antibodies or antigen binding portions thereof.

Description

Antibody against CCR2 {ANTIBODIES TO CCR2}

Cross Reference to Related Patents and Patent Applications

This application claims the benefit of US Provisional Application No. 61 / 189,357, filed August 18, 2008, which is incorporated herein by reference in its entirety.

A joint research agreement

The present disclosure and claims herein are a joint study between Pfizer Inc. and Apgenix Inc., which enter into force when or before the subject matter of the claimed invention is made. As a result of activities made within the scope of the contract.

Leukocyte infiltration into the site of inflammation is thought to be regulated by a 8-10 kD protein known as chemokine. The chemokines are classified into four groups called CC, CXC, XC and CX3C, depending on the spacing of their N terminal cysteine residues. Chemokines can mediate a series of proinflammatory effects on leukocytes such as chemotactic triggering, degranulation, lipid mediator synthesis and integrin activation (Oppenheim, JJ et al., Annu. Rev. Immunol., 9: 617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55: 97-179 (1994); Miller, MD and Krangel, MS, Crit. Rev. Immunol., 12: 17-46 (1992) ).

One chemokine, also known as CCL2, is a monocyte chemotactic protein-1 (MCP-1) that acts on monocytes, lymphocytes and dendritic cells, resulting in chemotaxis, granule release, respiratory blast and cytokine release. Induce. Research suggests that MCP-1 is involved in the pathology of diseases such as rheumatoid arthritis, atherosclerosis, granulomatous disease, chronic obstructive pulmonary disease (COPD), obesity / diabetes, neuropathic pain, cancer and multiple sclerosis (Koch, J. Clin. Invest. 90: 772-79 (1992); Hosaka et al., Clin.Exp. Immunol. 97: 451-457 (1994); Schwartz et al., Am. J. Cardiol. 71 (6): 9B-14B (1993); Schimmer et al., J. Immunol. 160: 1466-1471 (1998); Flory et al., Lab. Invest. 69: 396-404 (1993); Gong et al., J. Exp. Med. 186: 131-137 (1997); Salcedo et al. Blood 96 (1) 34-40 (2000); Bracke et al., Inflammation & Allergy-Drug Targets 6: 75-79 (2007); Chung Current Drug Targets-Inflammation & Allergy 4: 619-625 (2005)).

CCR2 is a chemotactic receptor bound to seven transmembrane domain G-proteins that bind to other chemokines including MCP-1 and CCL8 (MCP-2), CCL7 (MCP-3) and CCL13 (MCP-4) (Charo , IF, et al., Proc. Natl. Acad. Sci. USA 91: 2752-2756 (1994); Myers, SJ, et al., J. Biol. Chem. 270: 5786-5792 (1995); Gong et al., J. Biol Chem 272: 11682-11685 (1997); Garcia-Zepeda et al., J. Immunol. 157: 5613-5626 (1996)). CCR2 is also known as CMKBR2 and CKR2. CCR2A and CCR2B, two alternatively spliced forms of CCR2 with different C termini, are cloned (Wong et al (1997) J. Biol. Chem. 272: 1038-1045). In signal transduction studies, both CCR2A and CCR2B mediate agonist dependent calcium mobilization and adenylyl cyclase inhibition. CCR2 is expressed on monocytes, T cells and dendritic cells and interacts with chemokines secreted by endothelial cells, monocytes and synovial fibroblasts.

The biological role of CCR2 is investigated through the use of CCR2 knockout mice (Boring et al., J Clin Invest. 100 (10): 2552-61 (1997); Boring et al., Nature 394 (6696) : 894-7 (1998); De Paolo et al., J Immunol. 171 (7): 3560-7 (2003); Gaupp et al., Am J Pathol. 162 (1): 139-50 (2003)) . CCR2-/-mice have significant deficiencies in both delayed hypersensitivity reactions and production of Th1 cytokines and are generally less susceptible to experimental autoimmune encephalomyelitis (EAE). In addition to modulating the immune response, CCR2 is a cofactor for HIV (Connor et al., J. Exp. Med. 185: 621-628 (1997); Frade et al., J Clin Invest. 100 (3) 497-502 (1997).

Due to the involvement of MCP-1 and its receptor CCR2 in undesirable immune responses, CCR2 antagonists may be promising therapeutics. However, few CCR2 antagonists are described (see Ogilvie et al., Blood 97 (7): 1920-4 (2001)). Thus, there is a need for new and improved compositions that bind to CCR2 and block CCR2 signal transduction mediated by its ligands.

The present invention provides an isolated antibody or antigen binding portion thereof, which can specifically bind to CCR2, in particular human CCR2, and can act as a CCR2 antagonist, and a composition comprising said antibody or portion thereof. The present invention includes an antibody or antigen binding portion thereof that binds CCR2 at an epitope other than the N-terminal portion or the third loop of CCR2. The antibody may bind to the first and / or second extracellular loop of CCR2.

The present invention provides an antibody comprising (i) a heavy and / or light chain of the anti-CCR2 antibody, a variable domain thereof or an antigen binding portion thereof, or a nucleic acid molecule encoding them; And (ii) a pharmaceutically acceptable carrier. The composition may further comprise other ingredients such as therapeutic or diagnostic agents.

The invention also provides methods of diagnosis and treatment. Similarly, the present invention provides anti-CCR2 antibodies and portions thereof for the manufacture of a medicament for treating inflammatory and non-inflammatory diseases.

The present invention provides methods for recombinantly producing vectors and host cells comprising nucleic acid molecules, and polypeptides encoded by nucleic acid molecules. The invention also provides an isolated cell line that produces an anti-CCR2 antibody or antigen binding portion thereof.

1A, 1B and 1C are graphs showing binding of CCR2 antibodies to cells assessed by FACS analysis. Figure 1a is a graph showing the binding of ^{AF-488 (ALEXA FLUOR ® 488} , Invitrogen) conjugated CCR2 antibody 4.40 A68G S230P on human whole blood mononuclear cells compared to the KLH control antibody is evaluated by FACS analysis. 1B is a graph showing binding of AF-488 conjugated CCR2 antibody 4.40 A68G S230P to 300-19 cells expressing human CCR2 as assessed by FACS analysis. 1C is a graph showing binding of 4.40 A68G S230P antibodies at various concentrations to CCR2 transfected 300-19 cells detected by anti-human PE.
2 shows dose related binding of 4.40 A68G S230P antibody to CCR2 transfected 300-19 cells in saturation binding assay.
3 shows the ability of a 4.40 A68G S230P antibody to inhibit chemotaxis of THP-1 cells in response to CCR2 ligand MCP-1 without reacting to CCR1 / CCR5 ligand MIP-1a.
4 shows the ability of a 4.40 A68G S230P antibody to inhibit chemotaxis of primary human monocytes in response to MCP-1.
5 shows a plasmid map of retroviral vectors for expression of CCR1 / CCR2 chimeras.
6A and 6B show binding of 4.40 A68G S230P antibody to 300-19 cells expressing chimeric receptors consisting of only the extracellular first and second loops of CCR2 and the N-terminus and third loops of CCR1. 6C shows the saturation binding assay of 4.40 A68G S230P on chimeric receptor transfected 300-19 cells measured by FACS analysis.
7 (A) 300-19 control cells; (B) transfected 300-19 cells expressing full length CCR2; (C) transfected 300-19 cells expressing flag tagged (M1) MRRR chimera (N terminus of flag tagged CCR2 (M) to ensure receptor expression and loop region of CCR1 (R)); (D) Transfected 300 expressing flag tagged (M1) RRRM chimera (N terminus and first and second loops of flag tagged CCR1 (R) to ensure receptor expression and third loop of CCR2 (M)) -19 cells; And (E) transfected expressing flag tagged (M1) RMMR chimera (N terminus and third loop of CCR1 (R) flag tagged to ensure receptor expression and first and second loops of CCR2 (M)). Saturation binding assay (3 hour saturation curve) of 4.40 A68G S230P antibody against 300-19 cells.
FIG. 8 shows binding of the 4.40 A68G S230P antibody to either the loop 2 peptide region or loop 3 peptide region of CCR2 evaluated in the capture ELISA.
9 shows two graphs showing FACS immunostaining of 300-19 cells expressing recombinant CCR5 by either AF-488 conjugated 4.40.3 A68G S230P antibody (A panel) or anti-CCR5 antibody (B panel). To show.
10 shows inhibition of MCP-1 induced activity by 4.40 A68G S230P antibody assessed by calcium mobilization in CCR2 transfected 300-19 cells.
FIG. 11 shows MCP-3 induced chemotaxis inhibition of CCR2 transfected 300-19 cells by 4.40 A68G S230P antibody.
12 shows inhibition of human monocyte actin polymerization in response to MCP-1 in whole blood by 4.40 A68G S230P antibody.
FIG. 13 shows inhibition of monocyte actin polymerization in female cynomolgus monkeys in response to MCP-1 in whole blood by 4.40 A68G S230P antibody.
Figure 14 shows dose dependent inhibition of collagen 1 mRNA synthesis in hHSC cell line, LI90 by 4.40 A68G S230P antibody.
FIG. 15 shows dose dependent inhibition of pERK phosphorylation in human whole blood at 10 nM MCP-1 by 4.40 A68G S230P antibody.
FIG. 16 shows a decrease in plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) activity in human CCR2 nut-in mice with 4.40 A68G S230P antibody 24 hours after a single ConA injection. will be.
17A-17D show mismatches only for 4.22.3, 4.40.2, 4.39.3 and 4.9.2 antibody heavy and light chain variable regions (4.22.3, 4.40.2, 4.39.3 and 4.9.2 antibodies, respectively). Is observed) showing the alignment of the germline amino acid sequences of the heavy and light chain variable regions. CDRs are underlined and mismatched blank (s) are indicated with pound signs (#).

Definition and general technique

Unless defined otherwise herein, scientific and technical terms used herein have the meaning commonly understood by one of ordinary skill in the art. In addition, singular terms include plural and plural terms include singular unless the context otherwise requires. All publications and other references mentioned herein are incorporated by reference in their entirety. In general, the nomenclature and their techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

Methods and techniques are generally performed according to conventional methods well known in the art and unless otherwise indicated, as described in the various general, more specific references cited and described throughout this specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), all of which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly performed in the art or as described herein. The nomenclature and laboratory procedures and techniques used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical formulations, formulations and delivery, and patient care.

The following terms should be understood to have the following meanings, unless otherwise noted:

The term "polypeptide" includes polypeptide analogs of natural or artificial proteins, protein fragments and protein sequences. The polypeptide may be a monomer or a polymer.

The term “isolated protein”, “isolated polypeptide” or “isolated antibody” is not (1) by its origin or source of induction (1) related to the naturally related component involved in its original state, or (2) from the same species Is a protein, polypeptide or antibody that is removed, (3) expressed by cells from different species, or (4) not present in nature. Thus, a polypeptide synthesized in a cell system that is chemically synthesized or naturally derived from other cellular systems is "isolated" from its naturally related components. Naturally related components can be substantially removed by isolation using protein purification techniques that are also well known in the art.

Examples of isolated antibodies include anti-CCR2 antibodies synthesized in vitro by affinity purified anti-CCR2 antibodies, hybridomas or other cell lines using CCR2 or portions thereof and human anti-CCR2 antibodies derived from transgenic mice. have.

A protein or polypeptide is "substantially pure", "substantially homologous," or "substantially purified" when at least about 60% to 75% of the sample represents a single species of polypeptide. The polypeptide or protein may be a monomer or a multimer. Substantially pure polypeptide or protein typically comprises about 50%, 60%, 70%, 80% or 90% W / W, more typically about 95%, and at least 99% pure of a protein sample. Protein purity or homology can be exhibited by a number of means well known in the art, for example, by polyacrylamide gel electrophoresis of protein samples followed by visualization of a single polypeptide band upon gel staining with dyes well known in the art. . For certain purposes, higher resolutions can be provided using HPLC or by other means well known in the purification arts.

As used herein, the term “antibody analog” refers to an antibody comprising a segment having substantial identity with a portion of an amino acid sequence and having one or more of the following properties: (1) specific binding to CCR2 under suitable binding conditions , (2) the ability to inhibit one or more biological activities of CCR2. Typically, antibody analogs include conservative amino acid substitutions (or insertions or deletions) relative to the original sequence. Analogs are typically at least 20 or 25 amino acids long, at least 50, 60, 70, 80, 90, 100, 150 or 200 amino acids in length or more, usually the full length heavy chain or It can be as long as the light chain. In some cases one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen Antibody analogs with dog, 16 or 17 substitutions.

In some cases, amino acid substitutions for an anti-CCR2 antibody or antigen-binding portion thereof can (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, and (3) bind affinity to protein complex formation. , (4) add or remove glycosylation sites, and (5) confer or alter other physicochemical or functional properties of this analog, but still retain specific binding to CCR2. Analogs can include various mutants of sequences other than the peptide sequences that are commonly present. In sequences that are typically present in a portion of a polypeptide outside the intermolecular contact forming domain (s), for example, single or multiple amino acid substitutions may be made, such as conservative amino acid substitutions. Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence; For example, the replacement amino acid must not alter the antiparallel β sheets that make up the immunoglobulin binding domain that appears in the parent sequence or interfere with other types of secondary structures that identify the parent sequence. In general, glycine and proline should not be used in antiparallel β sheets. Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991); Thornton et al., Nature 354: 105 (1991), incorporated herein by reference). have.

When an “antibody” is mentioned herein, this generally means that the antigen binding portion thereof may also be used. The antigen binding moiety competes with an intact antibody for specific binding. In general, Fundamental Innunology, Ch. 7 (Paul, W., ed., Second ed. Raven Press, NY (1989)), incorporated by reference in its entirety for all purposes .. The antigen binding moiety is either by recombinant DNA techniques or intact. In some cases, antigen binding moieties include Fab, Fab ', F (ab') ₂ , Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chains. Polypeptides comprising at least a portion of an antibody (eg, scFv), a chimeric antibody, a binary antibody and an antibody sufficient to confer specific antigen binding to the polypeptide.

From the N terminus to the C terminus, both the mature light and heavy chain variable domains of the antibody comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions. The alignment of amino acids for each domain herein is described by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989).

As used herein, the antibody indicated by the number is the same as the monoclonal antibody obtained from the same number of hybridomas. For example, monoclonal antibody 4.40 is the same antibody as obtained from hybridoma 4.40 or a subclone thereof. Sequential subclones are referred to, for example, 4.40.1, 4.40.2 and 4.40.3, and have substantially the same sequence and function.

Fd fragment as used herein refers to an antibody fragment consisting of the V _H and C _H 1 domains; Fv fragments consist of the V _L and V _H domains of a single arm of an antibody; dAb fragment (Ward et al., Nature 341: 544-546 (1989)) consists of the V _H domain.

In some cases, the antibody is a single chain antibody (eg scFv) that forms a monovalent molecule via a synthetic linker that allows the V _L and V _H domains to be paired and made as a single polypeptide chain (eg, literature (Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). In some cases, an antibody uses two linkers, ie, V _H and V _L domains, expressed as a single polypeptide chain, but with a linker that is too short to pair between two domains on the same chain, so that the domain is another chain It is a bivalent antibody that is paired with the complementarity domain of and produces two antigen binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 ( 1993); Poljak RJ et al., Structure 2: 1121-1123 (1994). In some cases, one or more CDRs from an antibody herein can be incorporated into a molecule covalently or non-covalently to make an immunoconjugate that specifically binds to CCR2. In such cases, the CDR (s) may be incorporated as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or non-covalently incorporated. In the case of having more than one binding site, the binding sites may be the same or different from each other.

As used herein, the term "human antibody" refers to any antibody in which the variable and constant domain sequences are human sequences. The term includes antibodies which have sequences derived from human genes, but which are altered to remove, for example, cysteine which may reduce possible immunogenicity, increase affinity and cause unwanted folding. The term includes antibodies produced recombinantly in non-human cells that can confer glycosylation that is not characteristic for human cells. This antibody can be prepared in a variety of ways as described herein.

As used herein, the term “chimeric antibody” refers to an antibody comprising regions from two or more different antibodies. In one case, one or more CDRs of the chimeric antibody are derived from human anti-CCR2 antibodies. In another case, all of the CDRs are derived from human anti-CCR2 antibodies. In another case, CDRs from one or more human anti-CCR2 antibodies are combined in chimeric antibodies. For example, the chimeric antibody may comprise CDR1 derived from the light chain of the first human anti-CCR2 antibody, CDR2 derived from the light chain of the second human anti-CCR2 antibody, and CDR3 derived from the light chain of the third human anti-CCR2 antibody. The CDRs of can be derived from one or more other antiCCR2 antibodies. In addition, the framework region may be derived from one of the anti-CCR2 antibodies in which one or more CDRs are taken from one or more different human antibodies.

In some cases, the chimeric antibody is a humanized antiCCR2 antibody. Humanized anti-CCR2 antibodies comprise amino acid sequences of one or more framework regions and / or amino acid sequences derived from CDRs derived from at least a portion of the constant regions of one or more human anti-CCR2 antibodies and non-human antiCCR2 antibodies.

The teachings of this specification allow one of ordinary skill in the art to readily prepare fragments or analogs of antibodies or immunoglobulin molecules. Preferred amino acid and carboxy termini of the fragment or analog occur near the boundary of the functional domain.

As used herein, the term "surface plasmon resonance" refers to real-time detection of protein concentration changes in a biosensor matrix using, for example, the BIACORE ™ system (Pharmacia Biosensor AB (Uppsala Sweden and Piscataway, NJ)). It refers to an optical phenomenon capable of analyzing biospecific interactions. For further explanation, see Jonsson U. et al., Ann. Biol. Clin. 51: 19-26 (1993); Jonsson U. et al., Biotechniques 11: 620-627 (1991); Jonsson B. et al., J. Mol. Recognit. 8: 125-131 (1995); Johnsson B. et al., Anal. Biochem. 198: 268-277 (1991).

The term “K _D ” refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. An antibody is said to specifically bind an antigen when the dissociation constant is 1 mM or less, 100 nM or less, or 10 nM or less. In some cases, K _D is 1 pM to 500 pM. In other cases, K _D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

The term “epitope” includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitope determinants generally consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and generally have specific three-dimensional structural properties and specific charge properties. Epitopes may be "linear" or "conformational". In linear epitopes, all points of interaction between a protein and an interacting molecule (eg, an antibody) occur linearly along the protein's primary amino acid sequence. In conformational epitopes, interaction points occur over amino acid residues on proteins that are separated from each other. Once the desired epitope on the antigen is determined, for example, the techniques described herein can be used to form antibodies to that epitope. Alternatively, during the discovery process, antibody production and characterization may explain information about preferred epitopes. From this information, antibodies can be screened competitively for binding to the same epitope. The approach to accomplish this is to conduct competition and mutual competition studies to identify antibodies that compete with or compete with each other for binding to CCR2, for example antibodies compete for binding to antigen. A fast search procedure for “binding” antibodies based on their mutual competition is described in International Patent Application WO03 / 48731.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (second Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference.

As used herein, the term "polynucleotide" refers to a modified form of either ribonucleotide or deoxyribonucleotide, or any form of nucleotide, in the form of a polymer of nucleotides of at least 10 bases in length. The term includes single stranded and double stranded forms.

As used herein, the term "isolated polynucleotide" means that by its origin "isolated polynucleotide" does not relate to all or a portion of a polynucleotide in which "isolated polynucleotide" is found in nature, or ( 2) this refers to a polynucleotide of genome, cDNA or synthetic origin, or some combination thereof, that is operably linked to a polynucleotide that is not linked in nature, or (3) is not found in nature as part of a larger sequence.

The term "natural nucleotide" as used herein includes deoxyribonucleotides and ribonucleotides. As used herein, the term “modified nucleotide” includes nucleotides having a modified or substituted group of sugars and the like. The term "oligonucleotide linkage" as referred to herein refers to oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoroanilothioate, phosphor Aniladate, phosphoramidate, and the like. See, eg, La Planche et al., Nucl. Acids Res. 14: 9081 (1986); Stec et al., J. Am. Chem. Soc. 106: 6077 (1984); Stein et al., Nucl. Acids Res. 16: 3209 (1988); Zon et al., Anti-Cancer Drug Design 6: 539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); US Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990), the disclosures of which are incorporated herein by reference. Oligonucleotides may include a label for detection if desired.

“Operably linked” sequences include both expression control sequences adjacent to a gene of interest and expression control sequences that function across or near a regulatory gene of interest. As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to carry out the process of encoding the expressed and ligated sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; Effective RNA processing signals such as splicing and polyadenylation signals; Sequences that stabilize cytoplasmic mRNA; Sequences that enhance translation efficiency (ie, Kozak consensus sequence); Sequences that enhance protein stability; And, if desired, sequences that enhance protein secretion. The nature of this regulatory sequence depends on the host organism. In prokaryotes this control sequence generally comprises a promoter, ribosomal binding site and transcription termination sequence, and in eukaryotes this control sequence generally comprises a promoter and transcription termination sequence. The term "regulatory sequence" is intended to include, at a minimum, all components necessary for its presence in expression and processing, and may also include additional components which are advantageous in presence, such as leader sequences and fusion partner sequences.

As used herein, the term "vector" refers to a nucleic acid molecule that can carry another nucleic acid to which it is linked. In some cases, the vector is a circular double stranded piece of plasmid, ie, DNA into which additional DNA segments can be ligated. In some cases, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some cases, the vector can autonomously proliferate in the host cell into which it is introduced (eg, bacterial vectors and episomal mammalian vectors with bacterial origin of replication). In other cases, the vector (eg, a non-episomal mammalian vector) can be integrated into the host cell genome upon introduction into the host cell and thus replicate with the host genome. In addition, some vectors may direct the expression of genes to which they are operatively linked. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector").

As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector is introduced. “Recombinant host cell” and “host cell” are to be understood to mean the particular subject cell, as well as the progeny of this cell. Since some modifications occur in descendant generation due to either mutation or environmental influence, this progeny may, in fact, not be identical to the parent cell, but is still included within the scope of the term "host cell" as used herein.

The term “sequence identity (%)” in relation to a nucleotide sequence means a residue of two sequences that are identical when aligned for maximum relevance. There are a number of different algorithms known in the art that can be used to determine nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, a program from Wisconsin Package Version 10.0, Genetics Computer Group (GCG) (Madison, Wisconsin). For example, FASTA, including programs FASTA2 and FASTA3, provides the alignment and percent sequence identity of the highest overlapping region between query sequence and search sequence (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson , Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 (1998); hereby incorporated by reference. Included). Unless otherwise noted, default parameters for a particular program or algorithm are used. Using FASTA with its default parameters (letter size of 6 and the NOPAM factor to classify the matrix) or with Gap with its default parameters provided in GCG Version 6.1 (incorporated herein by reference), For example, percent sequence identity between nucleotide sequences can be determined.

Reference to nucleotide sequences includes the complements thereof unless otherwise noted. Thus, reference to a nucleic acid having a particular sequence should be understood to include its complementary strand along with its complementary sequence.

As used herein, the terms "sequence identity (%)" and "sequence homology (%)" are used interchangeably.

The term “substantial similarity” or “substantial sequence similarity”, when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or complementary strand thereof) with the appropriate nucleotide insertion or deletion, is described as FASTA, BLAST or Nucleotide sequence at at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as determined by any well-known algorithm of sequence identity such as Gap It means that identity exists.

When applied to a polypeptide, the term "substantial identity" refers to at least 70%, 75% or 80 when the two peptide sequences are optimally aligned using the default gap weight provided by the program, such as by program GAP or BESTFIT. It is meant to share% sequence identity, at least 90% or 95% sequence identity, and at least 97%, 98% or 99% sequence identity. In some cases, residue portions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity can be adjusted up to correct for the conservative nature of the substitution. Means of making such adjustments are well known to those skilled in the art. See, eg, Pearson, Methods Mol. Biol. 243: 307-31 (1994). Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) basic side chains: lysine, arginine and histidine; (6) acidic side chains: aspartic acid and glutamic acid; And (7) sulfur containing side chains: cysteine and methionine. Conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine.

Alternatively, conservative substitutions are any changes with positive values in the PAM250 algebraic matrix disclosed in Cornnet et al., Science 256: 1443-45 (1992), incorporated herein by reference. A "reasonably conservative" replacement is any change with non-negative values in the PAM250 algebraic matrix.

Sequence identity software is commonly used to determine sequence identity for polypeptides. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG can be programmed to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides of different species origin of an organism, or sequence homology or sequence identity between a wild-type protein and its mutane. Programs that can be used with default parameters specified by < RTI ID = 0.0 > See, for example, GCG Version 6.1 (University of Wisconsin, Wisconsin). Polypeptide sequences can also be compared using FASTA using default or recommended parameters, see GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provides the alignment and percent sequence identity of the highest overlapping region between query sequence and search sequence (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000)). Another preferred algorithm when comparing a sequence to a database containing numerous sequences of different organism origin is the computer program BLAST, in particular blastp or tblastn, using the default parameters provided by the program. See, eg, Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997).

CCR2 is a seven transmembrane domain protein, so it has six loops. Loop 1, loop 3 and loop 5 are intracellular loops when counting from the extracellular N terminus, while loop 2, loop 4 and loop 6 are extracellular loops. The first, second and third extracellular loops of CCR2 mean loop 2, loop 4 and loop 6, respectively.

Throughout this specification and claims, the phrase “comprises” or variations, such as “comprising”, is interpreted to imply the inclusion of the mentioned integers or groups of integers, but excludes the exclusion of any other integers or groups of integers. It is not to be interpreted as suggestive.

Human anti-CCR2 antibody and its identification

In some cases, the present invention provides human anti-CCR2 antibodies. In some cases, human anti-CCR2 antibodies are prepared by immunizing non-human transgenic animals, eg rodents, whose genomes include human immunoglobulin genes, so that the transgenic animals produce human antibodies. In some cases, the anti-CCR2 antibody and antigen binding moiety binds to (i) the first or second extracellular loop or both of CCR2; (Ii) does not bind to the N-terminal end or the third extracellular loop or both of CCR2; (Iii) include, but are not limited to, antibodies or antigen binding moieties that both (iii) and (ii) do. In another case, the present invention provides a human anti-CCR2 antibody that binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 128 or SEQ ID NO: 129. In another case, a human anti-CCR2 antibody binds to a polypeptide comprising an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 128 or SEQ ID NO: 129 do. In another case, the anti-CCR2 antibody and antigen binding moiety include, but are not limited to, an antibody or antigen binding moiety that binds to the third extracellular loop of CCR2.

The V _H , V _K , V _λ genes are classified into families based on sequence homology. Two V _H , V _K , V _λ genes belong to the same family when they share the same nucleotide sequence at> 80% positions. Anti-CCR2 antibodies may comprise human kappa light chain (V _K ) or human lambda light chain (V _λ ) or amino acid sequences derived therefrom. In some cases, including lambda light chains, the light chain variable domains (V _L ) are human V _λ 1, V _λ 2, V _λ 3, V _λ 4, V _λ 5, V _λ 6, V _λ 7, V _λ 8, V _λ 9 or V _λ 10 family genes (Williams SC et al. J. Mol. Bio. 264: 220-232 (1996)).

In some cases comprising a kappa light chain, the light chain variable domain (V _L) of the human V _K Ⅰ, V _K Ⅱ, V _K Ⅲ, V _K Ⅳ, V _K Ⅴ or V _K Ⅵ family gene (Cox JPL, et al. , Eur J. Immunol 24:. 827-836 (1994)), preferably from V ⅰ _K, V _K ⅱ, ⅳ V _K or V _K gene family ⅵ, preferably V _K or V ⅰ the _K gene family ⅵ I use it. In some cases, the light chain germline sequences are A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4 / 18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4 and Human V _K sequences, including but not limited to O8. In some cases, these light chain human germline genes are V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2- 7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4 and Selected from V5-6. In some cases, the light chain is a human V _K O12 Ⅰ, _K Ⅱ V A1, V _K V _K Ⅵ Ⅳ B3 or A26 uses the germline gene.

Anti-CCR2 antibodies may comprise heavy chain variable domains (V _H ) utilizing human V _H 1, V _H 2, V _H 3, V _H 4, V _H 5, V _H 6 or V _H 7 family genes. In certain instances, these heavy chain human germline genes are selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3- 38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1 and VH7-81. In some cases, the heavy chain of the human V _H or V _H 1-46 Ⅰ Use the III 3-30 gene.

In certain cases, the light chain variable region and / or heavy chain variable region comprise a framework region or at least a portion of the framework region (eg, comprise two or three subregions, such as FR2 and FR3). In some cases, at least FRL1, FRL2, FRL3 or FRL4 are fully human. In another example, at least FRH1, FRH2, FRH3 or FRH4 is fully human. In some cases, at least FRL1, FRL2, FRL3, or FRL4 are germline sequences (e.g., human germline) or human consensus sequences for a particular framework (easily available from a source of known human Ig sequences described herein). It includes. In another example, at least FRH1, FRH2, FRH3 or FRH4 is a germline sequence (eg, a human gonad) or comprises a human consensus sequence for a particular framework.

In some cases, the V _L of the CCR2 antibody comprises one or more amino acid substitutions, deletions and / or insertions relative to the germline amino acid sequence of the human gene. In some cases, the V _L of an antiCCR2 antibody comprises one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions relative to the germline amino acid sequence. . In some cases, one or more of these substitutions, deletions and / or insertions occurs in the CDRs of the light chain. In some cases, amino acid substitutions, deletions, and / or insertions into the gonads are one or more identical as substitutions, deletions and / or insertions into the gonads in any one or more V _L of antibody 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. Takes place in position. For example, anti-CCR2 antibodies of the V _L may comprise one or more amino acid substitution, deletion and / or insertion as compared to germline found in the V _L of the antibody 4.40. In some cases, amino acid alterations occur at one or more of the same positions, but include different substitutions, deletions, and / or insertions into the gonads. In some cases, substitutions may indicate conservative amino acid substitutions at this position (s) relative to amino acids in the reference antibody. For example, if one of these antibodies is substituted for the gonad and is glutamate, then aspartate can be substituted at that position. Similarly, if the amino acid substitution is serine compared to the gonad, then the position may be conservatively substituted for threonine for serine.

In some cases, the light chain of a human anti-CCR2 antibody is antibody 4.40 (SEQ ID NO: 101); 4.9 (SEQ ID NO: 29); 4.22 (SEQ ID NO: 65); 4.39 (SEQ ID NO: 194); Or the variable domain (V _L ) amino acid sequence of 4.40 A68G S230P (SEQ ID NO: 113); Or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions and / or no more than 3 non-conservative amino acid substitutions in total It includes the amino acid sequence having. In some cases, the light chains are CDR1, CDR2 and CDR3, or less than 4 or less than 3, respectively, selected independently from the light chain CDR1, CDR2 and CDR3 of the light chain of antibody 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P, respectively. CDRs having an amino acid substitution and / or a total of up to three non-conservative amino acid substitutions. In some cases, the light chains of the anti-CCR2 antibody are each monoclonal antibody 4.40 (SEQ ID NO: 100); 4.9 (SEQ ID NO: 28); 4.22 (SEQ ID NO: 64); 4.39 (SEQ ID NO: 193); Or light chain CDR1, CDR2 and CDR3 independently selected from light chain CDR1, CDR2 and CDR3 regions of 4.40 A68G S230P (SEQ ID NO: 112). In some cases, the light chain of the anti-CCR2 antibody is 4.40 (SEQ ID NO: 101); 4.9 (SEQ ID NO: 29); 4.22 (SEQ ID NO: 65); 4.39 (SEQ ID NO: 194); Or light chain CDR1, CDR2 and CDR3 of the antibody comprising the amino acid sequence of the V _L region of the antibody selected from 4.40 A68G S230P (SEQ ID NO: 113); Or said CDRs having less than 4 or less than 3 conservative amino acid substitutions and / or not more than 3 total non-conservative amino acid substitutions each. Sequence identifiers are described for the CDRs of some antibodies in Table 8.

With respect to the heavy chain, in some cases the variable domain (V _H ) uses human V _H 3-30 or V _H 1-46 gene sequences. In some cases, the V _H sequence of an antiCCR2 antibody includes one or more amino acid substitutions, deletions, and / or insertions (additions) relative to the germline amino acid sequence. In some cases, the variable domains of the heavy chains are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 from the germline amino acid sequence. Dogs, 14, 15, 16 or 17 substitutions, deletions and / or insertions. In some cases, the substitution is a non-conservative substitution compared to the germline amino acid sequence. In some cases, substitutions, deletions and / or insertions occur in the CDRs of the heavy chains. In some cases, amino acid substitutions, deletions and / or insertions occur at one or more identical positions as mutations from the germline at any one or more V _H of antibody 4.40, 4.22, 4.39 or 4.9. In other cases, amino acid substitutions, deletions and / or insertions occur at one or more of the same positions but include different substitutions, deletions and / or insertions than in the reference antibody. In some cases, the antibody comprises heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 202 or SEQ ID NO: 203.

In some cases, the heavy chain may comprise antibody 4.40 (SEQ ID NO: 83); 4.22 (SEQ ID NO: 47); 4.9 (SEQ ID NO: 11); The V _H amino acid sequence of 4.39 (SEQ ID NO: 176); Or said V _H amino acid sequence having up to 1, 2, 3, 4, 6, 8, or 10 conservative amino acid substitutions and / or up to 3 non-conservative amino acid substitutions . In some cases, the heavy chain comprises the amino acid sequence from the beginning of CDR1 to the end of CDR3 of any one of the antibodies.

In some cases, the heavy chain is a heavy chain CDR1, CDR2 and CDR3 of antibody 4.40, 4.22, 4.39 or 4.9 or up to 8, 6 or less, 4 or less, 3 or less conservative amino acid substitutions and / or 3 or less in total CDRs having non-conservative amino acid substitutions of

In some cases, the heavy chain CDRs are independently selected from the CDRs of antibody 4.40, 4.22, 4.39 or 4.9. In another case, the heavy chain was 4.40 (SEQ ID NO: 83); 4.22 (SEQ ID NO: 47); CDRs independently selected from two or more V _H regions selected from 4.39 (SEQ ID NO: 176) or 4.9 (SEQ ID NO: 11). In another case, the antibody comprises the light chain disclosed above and the heavy chain disclosed above. In further cases, the light chain CDRs and heavy chain CDRs are of the same antibody origin.

One type of amino acid substitution that can be made is to modify one or more cysteines in an antibody that can be chemically reactive to another residue, such as, but not limited to, alanine or serine. In one case, there is a substitution of unvariable cysteine. Substitutions can be made in the CDR or framework regions of the variable domains or in the constant domains of antibodies. In some cases, cysteine is a variable.

Another type of amino acid substitution that can be made is to alter any potential proteolytic site in the antibody. This site may occur in the CDR or framework regions of the variable domains or in the constant domains of antibodies. Substitution of cysteine residues and removal of proteolytic sites can reduce the risk of any heterogeneity in antibody products and thus increase their homogeneity. Another type of amino acid substitution is to remove asparagine-glycine pairs that alter one residue or both to form a potential deamidation site. In some cases, amino acid substitutions are used to insert or remove glycosylation sites. In some cases, the C-terminal lysine of the heavy chain of the anti-CCR2 antibody can be removed proteolytically or genetically. In various cases, the heavy and light chains of the anti-CCR2 antibody may optionally comprise a signal sequence.

In one aspect, antibodies are prepared by hybridomas.

Table 1 lists the sequence identifier (SEQ ID NO) of the nucleic acid encoding the full length and variable domain containing portions of the heavy and light chains and the corresponding analogous amino acid sequences of the exemplary antibodies.

TABLE 1

The invention also provides several heavy and / or light chain variants of the human anti-CCR2 antibodies described above comprising one or more amino acid substitutions. To designate a variant, the first spelling is the one word symbol for an amino acid in a naturally occurring antibody chain, the number indicating the position of the amino acid, where the first position is the N terminal amino acid, and the second spelling the variant One word sign for amino acids. In some cases, the present invention provides heavy chain variants. One such heavy chain variant is a hinge region that stabilizes mutations that reduce the formation of semimonomers (Angal, S. et al. Molecular Immunology 30: 105-108 (1993)). 4.40 One such hinge that stabilizes mutations in antibody heavy chain variants has a proline substitution for serine at position 230 of SEQ ID NO: 82. The DNA sequence encoding the S230P variant has a CCA codon starting at position 688 of SEQ ID NO: 115.

The present invention also provides variant light chains of monoclonal antibody 4.40. A68G is a 4.40 light chain variant represented by SEQ ID NO: 113 wherein residue 68 is a glycine residue. In the DNA sequence, the A68G 4.40 variant is encoded by SEQ ID NO: 109, wherein the codon starting at position 252 is GGG.

In other cases, antibodies can be prepared comprising a combination of amino acid variants. An example of a combination of variants is anti-CCR2 antibody 4.40 A68G S230P comprising light chain substitution A68G and heavy chain substitution S230P in connection with the 4.40 antibody. Further combinations of variant heavy chains and variant light chains of 4.40.

In one case, the anti-CCR2 antibody is 4.40, 4.22, 4.39, 4.40 A68G S230P or 4.9. In still further cases, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or 99 of the variable domain amino acid sequences of any of the human anti-CCR2 antibodies described above Antibody comprising a variable domain amino acid sequence having greater than% sequence identity.

Classes and Subclasses of Anti-CCR2 Antibodies

Classes and subclasses of anti-CCR2 antibodies can be determined by any method known in the art. In general, antibodies specific for a particular class and subclass of the antibody can be used to determine the class and subclass of the antibody. This antibody is commercially available. Classes and subclasses can be determined by ELISA or Western blot and other techniques. Alternatively, the amino acid sequence may be compared with known amino acid sequences of various classes of immunoglobulins to sequence all or a portion of the constant domains of the heavy and / or light chains of the antibody, and to determine the class and subclass of the antibody to determine the class and The subclass can be determined.

In some cases, the antiCCR2 antibody is a monoclonal antibody. The antiCCR2 antibody may be an IgG, IgM, IgE, IgA or IgD molecule. In one case, the anti-CCR2 antibody is, for example, IgG belonging to an IgG1, IgG2, IgG3 or IgG4 subclass. In another case, the antiCCR2 antibody is IgG4. In yet another case, the antibody is an IgG4 isoform (Ellison J. and Hood L., PNAS 79: 1984-1988 (1982); & Brusco A. et al., Eur J. Immunogenticsl 25: 349-355 (1998).

Binding Affinity of Anti-CCR2 Antibodies to CCR2

In some cases, anti-CCR2 antibodies bind to CCR2 with high affinity.

In some cases, the anti-CCR2 antibody binds with high affinity to the first extracellular loop of CCR2, to the second extracellular loop of CCR2, or to the epitope formed by both the first and second extracellular loops.

In related cases, the anti-CCR2 antibody binds to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 128 or SEQ ID NO: 129.

In some cases, the anti-CCR2 antibody does not bind to the third extracellular loop of CCR2 or the N terminal domain of CCR2.

In another case, the antiCCR2 antibody does not bind to the peptide consisting of the sequences set forth in SEQ ID NO: 127 or SEQ ID NO: 130. In another case, the anti-CCR2 antibody has about 2 × 10 ⁻⁷ M or less, about 2 × 10 ⁻⁸ M or less, about 2 × 10 ⁻⁹ M or less, about 1 × 10 ⁻⁹ M or less, about 9 × 10 ^{− 10} M or less, about 8 × 10 ^-10 M or less, about 7 × 10 ^-10 M or less, about 6 × 10 ^-10 M or less, 5 × 10 ^-10 M or less, about 4 × 10 ^-10 M or less, about 3 Binds to the first and / or second extracellular loop of CCR2 with a K _D of 10 × 10 ⁻¹⁰ M or less or about 2 × 10 ⁻¹⁰ M or less. In some cases, the antibody binds CCR2, or the first and / or second extracellular loop of CCR2 with a substantially identical K _D as an antibody selected from 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. Lot to another case, the antibody is SEQ ID NO: 83, SEQ ID NO: 11, with substantially the same K _D as CCR2 an antibody comprising a heavy chain variable domain having the amino acid sequence of V _H areas that are found in SEQ ID NO: 176 or SEQ ID NO: 47, Or binds to the first and / or second extracellular loop of CCR2. In yet another case, the antibody comprises the CDRs of a light chain variable domain having the amino acid sequence of a V _L region found in SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 29, SEQ ID NO: 194, or SEQ ID NO: 65, or SEQ ID NO: 83 , An antibody comprising the CDRs of a heavy chain variable domain having the amino acid sequence of the V _H region found in SEQ ID NO: 11, SEQ ID NO: 176 or SEQ ID NO: 47, binds CCR2 with a substantially identical K _D.

In some cases, anti-CCR2 antibodies may have a low dissociation rate constant (k _off ). In some cases, the anti-CCR2 antibody is directed to CCR2, more preferably CCR2, with k _off of 1.0 × 10 ⁻³ s ⁻¹ or less, 5.0 × 10 ⁻⁴ s ⁻¹ or less, or 2 × 10 ⁻⁴ s ⁻¹ or less. Binds to the first and / or second extracellular loop. In some cases, k _off can be substantially the same as the antibodies described herein, including antibodies selected from 4.40, 4.9, 4.22, 4.39 and 4.40 A68G S230P. In some cases, the antibody is an antibody comprising a CDR of a heavy chain or a CDR of a light chain from an antibody selected from 4.40, 4.9 and 4.40 A68G S230P, with substantially the same k _off first and / or second extracellular of CCR2, or CCR2 Can be coupled to a loop. In some cases, the antibody comprises (i) a heavy chain variable domain having the amino acid sequence of the V _H region found in SEQ ID NO: 83 or SEQ ID NO: 11, (ii) V _L found in SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 29 A light chain variable domain having an amino acid sequence of a region, or (iii) binding to CCR2, or the first and / or second extracellular loop of CCR2, with substantially the same k _off , with both k) and (ii) can do. In yet another instance, the antibody may comprise the CDRs of a light chain variable domain having the amino acid sequence of the V _L region found in SEQ ID NO: 101, SEQ ID NO: 113 or SEQ ID NO: 29; And a CDR of a heavy chain variable domain having the amino acid sequence of the V _H region found in SEQ ID NO: 83 or SEQ ID NO: 11, wherein the first and / or second extracellular loop of CCR2, or CCR2, is substantially the same k _off ; Can be combined.

The binding affinity and dissociation rate of anti-CCR2 antibodies to CCR2 can be determined by methods known in the art. Binding affinity can be measured by ELISA, RIA, flow cytometry, surface plasmon resonance, such as BIACORE ™. Dissociation rates can be measured by surface plasmon resonance. Methods known in the art can be used to determine whether an antibody has a K _D substantially the same as an antiCCR2 antibody. Example 5 illustrates a method for the crystal affinity constants of anti-CCR2 monoclonal antibodies.

Identification of CCR2 Epitopes Recognized by Anti-CCR2 Antibodies

The invention binds to CCR2 and comprises: (a) an antibody selected from 4.40, 4.9, 4.22, 4.39 and 4.40 A68G S230P; (b) an antibody comprising a heavy chain variable domain having the amino acid sequence of the variable domain found in SEQ ID NO: 83, SEQ ID NO: 11, SEQ ID NO: 176, or SEQ ID NO: 47, (c) SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 29 , An antibody comprising a light chain variable domain having the amino acid sequence of a variable domain found in SEQ ID NO: 194 or SEQ ID NO: 65, or (d) a heavy chain variable domain as defined in (b) and a light chain variable domain as defined in (c) Provided are human anti-CCR2 monoclonal antibodies capable of competing or competing with and / or binding to the same epitope as antibodies that comprise different. When two antibodies reciprocally compete for each other for binding to CCR2, it is said to compete with each other.

It is possible to determine whether an antibody binds to, competes with, or competes with the same epitope for binding to an anti-CCR2 antibody provided herein using methods known in the art. In one case, the anti-CCR2 antibodies provided herein are allowed to bind CCR2 under saturation conditions and then measure the ability of the test antibody to bind CCR2. If the test antibody binds to CCR2 at the same time that an anti-CCR2 antibody is provided, the test antibody binds to a different epitope as an anti-CCR2 antibody. However, if the test antibody is unable to bind CCR2 at the same time, the test antibody binds to an epitope in close proximity to the same epitope, overlapping epitope or epitope bound by human anti-CCR2 antibody provided herein. To determine if the test antibody competes with the reference antibody, the experiment is performed by reversing the antibody, ie, allowing the test antibody to bind CCR2 and then measuring the ability of the anti-CCR2 antibody provided herein to bind CCR2. This experiment can be performed using ELISA, RIA, BIACORE ™ or flow cytometry (FACS).

Inhibition of CCR2 Activity by Anti-CCR2 Antibodies

In some cases, the present invention provides anti-CCR2 antibodies that inhibit CCR2 mediated signal transduction. In other instances, the present invention provides anti-CCR2 antibodies that inhibit MCP-1, MCP-2, MCP-3 and / or MCP-4 mediated signal transduction through CCR2. In other instances, the present invention provides anti-CCR2 antibodies that inhibit the binding of MCP-1 MCP-2, MCP-3 and / or MCP-4 to CCR2. In one case, CCR2 is human CCR2. In some cases, CCR2 is human CCR2A, CCR2B or both. In yet another case, the anti-CCR2 antibody is a human antibody.

IC ₅₀ of anti-CCR2 antibodies can be measured in ligand binding assays such as ELISA, RIA or related assays and cell based assays such as chemotaxis assays of cells expressing CCR2. In various cases, the antibody or antigen-binding portion thereof is MCP-1 and CCR2 with an IC ₅₀ of 5 μg / ml or less, 1 μg / ml or less, 0.5 μg / ml or less or 0.20 μg / ml or less as determined by ELISA assay. Inhibits ligand binding between

In another case, the present invention provides anti-CCR2 antibodies that reduce the activation of CCR2 in the presence of CCR2 ligands such as MCP-1 (CCL2), MCP-2, MCP-3 and / or MCP-4. In one case, the anti-CCR2 antibody comprises CCR2 ligand induced (i) G-protein activation, (ii) adenylate cyclase activation, (i) mitogen activating protein kinase (MAPK) activation, (iv) cytosolic calcium Mobilization, (v) ERK phosphorylation, (vi) chemotaxis or (vii) actin polymerization. Anti-CCR2 antibodies determine the GTP / GDP ratio of G-proteins in cells labeled with radiolabeled GTP, measure GTPgS integration, measure cytosolic calcium influx using calcium chromophores or phosphorylation status of MAPK in cells Can be measured to prevent, inhibit or reduce the activation of CCR2 in the presence of MCP-1. Assays for detecting CCR2 activation and / or MCP-1 binding to CCR2 are described, for example, in Gabrilin et al., Biochem Biophys Res Commun. 327 (2): 533-40 (2005); Jimenez-Sainz et al., Mol Pharmacol. 64 (3): 773-82 (2003).

In one case, chemotaxis assays can be used to determine the level of CCR2 activation. In some cases, the IC ₅₀ measured using the chemotaxis assay is 5 μg / ml or less, 1 μg / ml or less, 0.5 μg / ml or less, or 0.20 μg / ml or less. Example 10 illustrates one type of assay that monitors calcium mobilization to measure inhibition of CCR2 by anti-CCR2 antibodies.

In another embodiment, cells can be contacted with an antibody to down regulate cell surface CCR2 expression after incubation with the antibody. In some cases, the incubation may be a short time (eg 4 hours) or a long time (eg 24 hours). Western blotting, ELISA or FACS analysis can be used to measure down regulation of cell surface CCR2 expression. In certain cases, cells may be contacted with antibodies to reduce cell surface CCR2 expression by at least 6%, at least 10%, at least 20%, at least 30% or at least 50%, as measured by western blotting or ELISA.

In another embodiment, the antibody reduces MCP-1 induced pERK phosphorylation. Western blotting, ELISA or FACS analysis can be used to measure down regulation of MCP-1 induced pERK phosphorylation. In certain cases, the antibody reduces at least 6%, at least 10%, at least 20%, at least 30% or at least 50% of the MCP-1 induced pERK phosphorylation measured by FACS analysis.

In vivo chemotaxis inhibition by anti-CCR2 antibody

In some cases, the present invention provides anti-CCR2 antibodies that inhibit chemotaxis of immune cells in vivo. Immune cells that are inhibited chemotaxis include peripheral blood mononuclear cells, THP cells, monocytes, memory T lymphocytes, dendritic cells, eosinophils, natural killer cells, and CCR2 + cells that have metastasized to adoption. In one case, the anti-CCR2 antibody inhibits immune cell chemotaxis in response to one or more MCP-1, MCP-2, MCP-3 and MCP-4. In one case, the chemokine is MCP-1. In yet another case, the chemokine is MCP-3. Anti-CCR2 antibodies can inhibit chemotaxis at sites of inflammation or injury.

In some cases, the invention also relates to fibroblast-like synoviocytes (FLS) (see Garcia-Vicuna et al., Arthritis Rheum. 50 (12): 3866-77 (2004)), Adult neural stem cells (see Widera et al., Eur J Cell Biol. 83 (8): 381-7 (2004)) and human fetal astrocytes (Andjelkovic et al., J Neurosci Res. 70 ( 2): 219-31 (2002)), which provide anti-CCR2 antibodies that inhibit chemotaxis of non-immune cells.

In one case, the antibody inhibits cell chemotaxis compared to the chemotaxis of cells in untreated animals. In another case, the anti-CCR2 antibody has chemotaxis of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 Inhibit%, 95% or 100%. In one case, chemotaxis inhibition is measured at least 1 hour after the animal has started treatment with the antibody. In another case, chemotaxis inhibition is measured at least 7 days after the animal has started treatment with the antibody. In another case, the anti-CCR2 antibody inhibits at least 10% to 100% chemotaxis.

Species and Molecular Selectivity

In another embodiment, the anti-CCR2 antibody exhibits both species and molecular selectivity. In some cases, anti-CCR2 antibodies bind to human and cynomolgus CCR2. Anti-CCR2 antibodies may bind to additional CCR2 of nonhuman primate species. In some cases, the anti-CCR2 antibody does not bind to mouse or rat CCR2. In accordance with the teachings of the specification, species selectivity for anti-CCR2 antibodies can be determined using methods well known in the art. For example, western blot, flow cytometry, ELISA, immunoprecipitation or RIA can be used to determine species selectivity. In one case, flow cytometry can be used to determine species selectivity. In another case, species specificity can be determined by assessing the ability of the antibody to inhibit the MCP-1 functional response using cells from that species. Examples thereof include chemotaxis, actin polymerization, and calcium mobilization.

In another case, the anti-CCR2 antibody has selectivity for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 126 (human CCR2B) relative to the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 131 (human CCR5). In another case, the anti-CCR2 antibody has selectivity for CCR2 for at least 2, at least 5, at least 10, at least 25, at least 50, or at least 100 times CCR5. In another case, a human anti-CCR2 antibody binds to a polypeptide comprising an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 126.

In another case, the anti-CCR2 antibody comprises at least 2, at least 5, at least 10, at least 25, at least 50 or at least 100 times the polypeptide for a polypeptide consisting of the amino acid sequence of SEQ ID NO: 131 (human CCR5). Selectivity for the polypeptide consisting of the amino acid sequence set forth in No. 125 (human CCR2A). In another case, a human anti-CCR2 antibody binds to a polypeptide comprising an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125.

The teachings of the specification can determine the selectivity of anti-CCR2 antibodies against CCR2 using methods well known in the art. For example, selectivity can be determined using Western blot, flow cytometry, ELISA, immunoprecipitation or RIA and / or functional assays such as chemotaxis, calcium recruitment or actin polymerization.

Methods of Producing Antibodies and Antibodies Producing Cell Lines

Immunization

In some cases, non-human transgenic animals comprising some or all of the human immunoglobulin heavy and light chain loci in the genome are immunized with the CCR2 antigen to prepare human antibodies. In one case, the non-human animal is a XENOMOUSE ™ animal (Amgen Fremont, Inc. (formerly Abgenix, Inc.) (Fremont, CA)).

XENOMOUSE ™ mice are engineered mouse strains that contain large fragments of human immunoglobulin heavy and light chain loci and lack mouse antibody production. See, for example, Green et al., Nature Genetics 7: 13-21 (1994) and US Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, See 6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, See WO 99/45031, WO 99/53049, WO 00/09560 and WO 00/037504.

In another aspect, the present invention provides a method for preparing anti-CCR2 antibodies from non-human non-mouse animals by immunizing a non-human transgenic animal comprising a human immunoglobulin locus with a CCR2 antigen. This animal can be produced using the methods described in the documents cited above. The methods disclosed in this document can be modified as described in US Pat. No. 5,994,619, which is incorporated herein by reference. US Pat. No. 5,994,619 describes methods for producing newly cultured inner cell mass (CICM) cells and cell lines derived from pigs and cattle, and transformed CICM cells with heterologous DNA inserted. CICM transformed cells can be used to generate cloned transgenic embryos, fetuses and offspring. The 5,994,619 patent also describes a method for producing a transgenic animal capable of delivering heterologous DNA to its offspring. In a preferred case, the non-human animal is a mammal, in particular a rat, sheep, pig, goat, cow or horse.

XENOMOUSE ™ mice produce an adult-like human repertoire of fully human antibodies and produce antigen specific human antibodies. In some cases, XENOMOUSE ™ mice comprise approximately 80% of the human antibody V gene repertoire through the introduction of germline placement fragments of the human heavy chain locus and the kappa light chain locus. In other cases, XENOMOUSE ™ mice further comprise approximately all of the human lambda light chain locus. Mendez et al., Nature Genetics 15: 146-156 (1997), Green and Jakobovits, J. Exp. Med. 188: 483-495 (1998) and WO 98/24893, the disclosures of which are incorporated herein by reference.

In another case, antibodies are generated using VELOCIMOUSE ™ technology (Regeneron Pharmaceuticals, Terrytown, NY) for immediate production of genetically modified mice directly from modified embryonic stem (ES) cells (Poueymirou WT). , et al., Nature Biotechnology 25: 91-99 (2007)).

In some cases, a nonhuman animal comprising a human immunoglobulin gene is an animal with a human immunoglobulin “minigene locus”. In the minigenic locus approach, the exogenous Ig locus is mimicked through the insertion of individual genes of Ig locus origin. Thus, constructs for insertion into animals form one or more V _H genes, one or more D _H genes, one or more J _H genes, a mu constant domain and a second constant domain (preferably a gamma constant domain). This approach is specifically described in US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205. 5,721,367, 5,789,215 and 5,643,763, which are incorporated herein by reference.

In another aspect, the invention provides a method of making a humanized anti-CCR2 antibody. In some cases, non-human animals are immunized with CCR2 antigens as described herein under conditions that allow antibody production. Antibody producing cells are isolated from animals and nucleic acids encoding the heavy and light chains of the antiCCR2 antibody of interest are isolated. This nucleic acid is subsequently engineered using techniques known to those skilled in the art and as described further below to reduce the amount of non-human sequences, ie, to humanize antibodies to reduce the immune response in humans.

In some cases, the CCR2 antigen is isolated and / or purified CCR2. In one case, the CCR2 antigen is human CCR2. In some cases, the CCR2 antigen is a fragment of CCR2. In some cases, the CCR2 fragment is an extracellular loop, N terminal domain, or C terminal end of CCR2. In some cases, the CCR2 fragment comprises a first or second extracellular loop of CCR2. In other cases, the CCR2 fragment comprises the amino acid sequence set forth in SEQ ID NO: 128 or SEQ ID NO: 129.

In other cases, the CCR2 fragment does not comprise a third extracellular loop or N terminal domain of CCR2.

In other cases, the CCR2 fragment does not comprise the amino acid sequence set forth in SEQ ID NO: 127 or SEQ ID NO: 130. In some cases, the CCR2 fragment comprises one or more epitopes of CCR2. In other cases, the CCR2 antigen is a cell that expresses or overexpresses CCR2 or an immunogenic fragment thereof on its surface. In some cases, the CCR2 antigen is a CCR2 fusion protein. In some cases, CCR2 is a synthetic peptide immunogen.

Animals can be immunized by any method known in the art. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods of immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, eg, Harlow and Lane, supra, and US Pat. No. 5,994,619. In one case, the CCR2 antigen is administered with an adjuvant that stimulates an immune response. Examples of adjuvant include full or incomplete Freud adjuvant, RIBI (muramil dipeptide) or ISCOM (immunostimulatory complex). The adjuvant may contain it in local deposits to prevent rapid spread of the polypeptide or may include substances that stimulate the host to secrete factors chemotactic for macrophages and other components of the immune system. When administering a polypeptide, the immunization schedule involves the administration of two or more of the polypeptides over several weeks. Example 1 illustrates a method for generating anti-CCR2 monoclonal antibodies in XENOMOUSE ™ mice.

Antibody Production and Antibody Production Cell Lines

After immunizing the animal with the CCR2 antigen, antibodies and / or antibody producing cells can be obtained from the animal. In some cases, animals are bled or sacrificed to obtain anti-CCR2 antibody containing serum from these animals. Serum can be used as is obtained from this animal, immunoglobulin fragments can be obtained from the serum, or anti-CCR2 antibodies can be purified from the serum.

In some cases, antibody producing immortalized cell lines are prepared from cells isolated from immunized animals. After immunization, the animal is sacrificed and the peripheral blood, lymph nodes and / or spleen B cells are immortalized by any means known in the art. Methods of immortalizing a cell include transfecting it with an oncogene, infecting it with an oncogene virus and culturing it under conditions selected for immortalizing cells, treating it with a carcinogenic or mutant compound, immortalizing it Fusion with cells, such as myeloma cells, and inactivation of tumor suppressor genes, but are not limited to these. See, eg, Harlow and Lane, supra. When fusion with myeloma cells is used, myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Immortalized cells are screened using CCR2, a portion thereof or cells expressing CCR2. In one case, the CCR2 moiety comprises (i) a first and / or second extracellular loop of CCR2; (Ii) comprises the amino acid sequence set forth in SEQ ID NO: 128 and / or SEQ ID NO: 129; (Iii) does not comprise a third extracellular loop and / or an N terminal domain of CCR2; (iv) does not comprise the amino acid sequence set forth in SEQ ID NO: 127 and / or SEQ ID NO: 130; (v) combinations thereof. In one case, initial screening is performed using enzyme-linked immunoassay (ELISA) or radioimmunoassay. Examples of ELISA screening are provided in WO 00/37504, which is incorporated herein by reference.

Anti-CCR2 antibody producing cells, such as hybridomas, are selected, cloned, and further screened for desirable properties, including robust growth, high antibody production, and desirable antibody properties, as described further below. In vivo syngeneic animals, hybridomas can be expanded in animals lacking the immune system, for example in nude mice or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those skilled in the art.

In one aspect, the immunized animal is a non-human animal that expresses a human immunoglobulin gene, and the spleen B cells are fused to a myeloma cell line of the same species origin as the non-human animal. In an exemplary case, the immunized animal is a XENOMOUSE ™ mouse and the myeloma cell line is a non-secreted mouse myeloma. In one case, the myeloma cell line is P3-X63-Ag8.653 (American Type Culture Collection). See, eg, Example 1.

Immortalization identifying the antibody associated with the first and / or second extracellular loop of CCR2 by testing whether the antibody is produced by a cell that binds to a peptide comprising the amino acid sequence of the first or second extracellular loop of CCR2. Screening of antibody producing cells can be accomplished. Alternatively or in combination, one may test antibodies produced by cells for binding to a chimeric chemokine receptor having the sequence of the first and / or second extracellular loop of CCR2 but predominantly having the sequence of another chemokine receptor. have. See Example 8, which illustrates the use of chimeras to map epitopes of anti-CCR2 antibodies. In the case of complementarity, it has mainly the sequence of CCR2 but lacks the wild type first and / or second extracellular loop of CCR2, for example having a first and / or second extracellular loop from another cytokine receptor, Antibodies produced by cells known to bind CCR2 can be tested for binding to chimeric chemokine receptors containing mutations in one or both of these extracellular loops.

In another aspect, the invention provides cells and cell lines (including hybridomas) that produce human anti-CCR2 antibodies. In one case, human anti-CCR2 antibodies produced by cells, cell lines or hybridomas are antagonists of CCR2. In yet another case, the human anti-CCR2 antibody binds to (i) the first and / or second extracellular loop of CCR2; (Ii) binds to the amino acid sequence set forth in SEQ ID NO: 128 and / or SEQ ID NO: 129; (Iii) does not bind to the third extracellular loop and / or the N terminal domain of CCR2; (iv) does not bind the amino acid sequence set forth in SEQ ID NO: 127 and / or SEQ ID NO: 130; (v) combinations thereof. In another case, human anti-CCR2 antibodies produced by cells, cell lines or hybridomas do not bind to the third extracellular loop with high affinity, do not bind to the N-terminal domain of CCR2 with high affinity, or with high affinity. Do not combine anywhere.

In yet another embodiment, the transgenic animal is immunized with CCR2, primary cells, such as spleen or peripheral blood cells, isolated from the immunized transgenic animal and identified individual cells that produce antibodies specific for the desired antigen. do. For example, a sense primer that isolates polyadenylation mRNA from each individual cell and anneals the variable region sequence, eg, a degenerate primer that recognizes most or all of the FR1 region of the human heavy and light chain variable region genes. And reverse transcriptase polymerase chain reaction (RT-PCR) using antisense primers that anneal variable or binding (J) region sequences. The cDNAs of the heavy and light chain variable domains are then cloned and expressed in any suitable host cell, for example myeloma cells, as chimeric antibodies with respective immunoglobulin constant regions, such as heavy and κ or λ constant domains. Babcook, J.S. et al., Proc. Natl. Acad. Sci. USA 93: 7843-48 (1996), incorporated herein by reference. Thereafter, anti-CCR2 antibodies can be identified and isolated as described herein.

In another aspect, phage display techniques can be used to provide a library comprising a repertoire of antibodies having varying affinity for CCR2. Primary B cells can be used directly as a source of DNA. For example, expression libraries, e.g. Phage display libraries transfected with E. coli can be prepared. The resulting cells are tested for immunoreactivity against CCR2. Techniques for identifying high affinity human antibodies from this library are described in Griffiths et al., EMBO J. 13: 3245-3260 (1994); Nissim et al., Ibid, pp. 692-698; Griffiths et al., Ibid, 12: 725-734, incorporated by reference. Ultimately, clones obtained from libraries that produce the desired amount of binding affinity for the antigen are identified and DNA encoding the product responsible for this binding is engineered for standard recombinant expression. Phage display libraries are also constructed using previously engineered nucleotide sequences and screened in a similar manner. In general, cDNAs encoding heavy and light chains are fed or linked independently to form Fv analogs for generation in phage libraries.

Phage libraries are then screened for antibodies with the highest affinity for CCR2 and genetic material recovered from appropriate clones. Additional screening times can increase the affinity of the original antibody isolated.

Recombinant Methods to Prepare Nucleic Acids, Vectors, Host Cells, and Antibodies

Nucleic acid

The invention also includes nucleic acid molecules encoding anti-CCR2 antibodies or antigen binding portions thereof. In some cases, different nucleic acid molecules encode heavy and light chains of anti-CCR2 immunoglobulins. In other cases, the same nucleic acid molecule encodes the heavy and light chains of anti-CCR2 immunoglobulins. In one case, the nucleic acid encodes a CCR2 antibody or antigen binding portion thereof.

In some cases, the nucleic acid molecule encoding the variable domain of the light chain (V _L ) is either JK1, JK2, JK4, or JK5 with or without mutations from human VK1, VK2, VK3, VK4, VK5 or VK6 gene family segments and gonads. Gene segments.

In some cases, the nucleic acid molecule encoding the light chain comprises one, two, three, four, five, six, seven, eight, nine or ten substitutions from the germline amino acid sequence (s). Encodes an amino acid sequence. In some cases, nucleic acid molecules have one, two, three, four, five, six, seven, eight, nine, or ten conservative amino acid substitutions compared to the germline V _K and J _K sequences. And / or a nucleotide sequence encoding a V _L amino acid sequence comprising one, two or three non-conservative substitutions. Substitutions in CDRs, framework regions or constant domains.

In some cases, the nucleic acid molecule encodes a V _L amino acid sequence comprising one or more variants compared to the germline sequence that is identical to the modification found in V _L of one of antibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. In some cases, a nucleic acid comprises a mutation at one or more of the same positions as a mutation from the germline in a CCR2 antibody provided herein, but with a different substitution, in some cases a conservative substitution as compared to a substitution in a given antibody. _{Encode the L} amino acid sequence.

In some cases, the nucleic acid molecule encodes at least three amino acid substitutions as compared to the germline sequence found in the V _L of one of antibodies 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P.

In some cases, the nucleic acid molecule is a V _L amino acid of monoclonal antibody 4.40 (SEQ ID NO: 101), 4.9 (SEQ ID NO: 29), 4.22 (SEQ ID NO: 65), 4.39 (SEQ ID NO: 194), or 4.40 A68G S230P (SEQ ID NO: 113). A nucleotide sequence encoding a sequence, or variant or portion thereof. In some cases, the nucleic acid encodes an amino acid sequence comprising the light chain CDRs of one of the antibodies described above. In some cases, the portion is a contiguous portion comprising CDR1-CDR3.

In some cases, the nucleic acid molecules are antibodies 4.40, 4.9, 4.22. 4.39 or 4.40 A68G a V _L amino acid sequence or SEQ ID NO: Any of the V _L zone S230P 101, SEQ ID NO: 113, SEQ ID NO: 29, SEQ ID NO: 194 or SEQ ID NO: of any one of the V _L region of the of the 65 amino acid sequence Encode a V _L amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical. Nucleic acid molecules include nucleic acids encoding the V _L regions found in SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 29, or SEQ ID NO: 65.

In another case, the nucleic acid comprises the full length light chain of an antibody selected from 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P or the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 112, SEQ ID NO: 64, SEQ ID NO: 193, or SEQ ID NO: 28 To a light chain, or to said light chain sequence comprising a mutation, such as those disclosed herein.

In yet another case, the nucleic acid molecule encodes a variable domain of heavy chain (V _H ) comprising a human 3-30 or 1-46 V _H gene sequence or sequence derived therefrom. In various cases, nucleic acid molecules include human 3-30 V _H gene sequences, human D1-7 gene sequences, and human J _H 3B gene sequences; Human 1-46 V _H gene sequence, human D1-7 gene sequence, and human J _H 3B gene sequence; Or using sequences derived from human genes.

In some cases, the nucleic acid molecule is one, two, three, four, five, six, seven, eight, nine, ten compared to the germline amino acid sequence of the human V, D, or J gene. Encode an amino acid sequence comprising 11, 12, 13, 14, 15, 16, 17 or 18 mutations. In some cases, the mutation is in the V _H region. In some cases, the mutation is in the CDRs.

In some cases, the nucleic acid molecule encodes an amino acid sequence comprising one or more amino acid mutations compared to the germline sequence identical to the amino acid mutations found in V _H of monoclonal antibody 4.40, 4.22, 4.39 or 4.9. In some cases, the nucleic acid encodes at least three amino acid mutations compared to the germline sequence identical to at least three amino acid mutations found in one of the monoclonal antibodies described above.

In some cases, the nucleic acid molecule encodes at least a portion of the V _H amino acid sequence of a monoclonal antibody selected from 4.40 (SEQ ID NO: 83), 4.22 (SEQ ID NO: 47), 4.39 (SEQ ID NO: 176), or 4.9 (SEQ ID NO: 11). Nucleotide sequences, variants thereof, or conservative amino acid mutations and / or such sequences with up to three non-conservative amino acid substitutions in total. In various cases, the sequence encodes one or more CDRs, CDR3 regions, all three CDRs, contiguous portions or the entire V _H region, with or without a signal sequence.

In some cases, a nucleic acid molecule comprises said sequence having a nucleotide sequence or signal sequence encoding an amino acid sequence of one of SEQ ID NO: 82, SEQ ID NO: 46, SEQ ID NO: 10, SEQ ID NO: 175, or SEQ ID NO: 116. In some preferred cases, the nucleic acid molecule comprises at least a portion of the nucleotide sequence of SEQ ID NO: 73 or SEQ ID NO: 115 or said sequence having a signal sequence. In some cases, the portion encodes a contiguous region, including a V _H region, a CDR3 region, all three CDRs or CDR1-CDR3 (with or without a signal sequence).

In some cases, the nucleic acid molecule is a V _H amino acid sequence of any one of the V _H regions of antibody 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P or SEQ ID NO: 83, SEQ ID NO: 47, SEQ ID NO: 176, or SEQ ID NO: 11 the coding of the one of at least 70% to the amino acid sequence of the V _H zones, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical amino acid sequences V _H. The invention also encodes an amino acid sequence of 4.40 (SEQ ID NO: 83), 4.22 (SEQ ID NO: 47), 4.39 (SEQ ID NO: 176) or 4.9 (SEQ ID NO: 11) or a V _H region thereof, or SEQ ID NO: 74, SEQ ID NO: 38 , Nucleic acids having the nucleotide sequence of SEQ ID NO: 167, SEQ ID NO: 2 or encoding a V _H region thereof.

In another case, the nucleic acid is the full length heavy chain of an antibody selected from 4.40, 4.22, 4.9, 4.39 or 4.40 A68G S230P, or with or without signal sequence SEQ ID NO: 10, SEQ ID NO: 46, SEQ ID NO: 82, SEQ ID NO: 175, or sequence A heavy chain having the amino acid sequence of number 116, or a heavy chain comprising a mutation, such as one of the variants described herein. In addition, the nucleic acid may comprise a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 37, SEQ ID NO: 73, SEQ ID NO: 166, or SEQ ID NO: 115, or a nucleic acid molecule encoding a heavy chain comprising a mutation, such as one of the variants described herein Can be.

Nucleic acid molecules encoding the heavy or light chains or portions thereof of anti-CCR2 antibodies can be isolated from any source producing this antibody. In various cases, nucleic acid molecules are isolated from B cells isolated from animals immunized with CCR2 or from immortalized cells derived from these B cells expressing anti-CCR2 antibodies. Methods for isolating mRNA encoding antibodies are well known in the art. See, eg, Sambrook et al. mRNA can be used to generate cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In one case, nucleic acid molecules are isolated from hybridomas with human immunoglobulin producing cells of nonhuman transgenic animal origin as one of the fusion partners. In other cases, human immunoglobulin producing cells are isolated from XENOMOUSE ™ animals. In another case, human immunoglobulin producing cells are of nonhuman non-mouse transgenic animal origin as described herein. In another case, nucleic acids are isolated from nonhuman transgenic animals. For example, nucleic acid molecules isolated from nonhuman transgenic animals can be used for humanized antibodies.

In some cases, the nucleic acid encoding the heavy chain of an antiCCR2 antibody comprises a nucleotide sequence encoding a V _H domain linked in-frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule encoding a light chain of an anti-CCR2 antibody may comprise a nucleotide sequence encoding a V _L domain in-frame linked to a nucleotide sequence encoding a light chain constant domain from any source.

In a further embodiment, the nucleic acid molecules encoding the variable domains of the heavy chain (V _H ) and / or light chain (V _L ) are “converted” to full length antibody genes. In one case, the V _H segment is transformed by inserting a heavy chain constant (C _H ) or light chain constant (C _L ) domain into an expression vector that already encodes, thereby converting a nucleic acid molecule encoding a V _H or V _L domain to a full length antibody gene. Is operably linked to the C _H segment (s) in the vector and / or the V _L segment is operably linked to the C _L segment in the vector. In the other case, V _H and / or V _L connecting the nucleic acid molecule encoding a domain using standard molecular biological techniques on the nucleic acid molecule encoding C _H and / or a C _L domain, for ligation of the V _H and g. And / or convert the nucleic acid molecule encoding the V _L domain into a full length antibody gene. Nucleotide sequences of human heavy and light chain immunoglobulin constant domain genes are known in the art. See, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991. Nucleic acid molecules encoding full-length heavy and / or light chains can then be expressed from cells into which it has been introduced and from which anti-CCR2 antibodies have been isolated.

Nucleic acid molecules can be used to recombinantly express anti-CCR2 antibodies. Nucleic acid molecules can also be used to generate chimeric antibodies, bispecific antibodies, single chain antibodies, immunoconjugates, binary antibodies, mutated antibodies and antibody derivatives as further described below. If the nucleic acid molecule is derived from a nonhuman transgenic animal, the nucleic acid molecule can also be used for antibody humanization as described herein.

In some cases, the present invention provides nucleic acids that encode a full-length heavy or full-length light chain, wherein the nucleotide sequence encoding the constant region amino acid sequence comprises one or more mutations as compared to the germline human constant region sequence. For example, nucleic acids can encode amino acid substitutions that improve the properties of the antibody by adding or removing glycosylation sites or by encoding substitutions that improve the stability or half-life of the antibody. Nucleic acids may also include “silent” mutations to add or remove restriction enzyme sites, eg, to facilitate cloning of the nucleic acid with a particular expression vector.

vector

The present invention provides vectors comprising nucleic acid molecules encoding the heavy and / or light chains or antigen binding portions thereof of the anti-CCR2 antibody. The invention also provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments and probes thereof.

In some cases, an anti-CCR2 wherein DNA encoding a partial or full-length light and / or heavy chain obtained as described herein is inserted into an expression vector such that the gene is operably linked to essential expression control sequences, such as transcriptional and translational control sequences. Express the antibody or antigen binding moiety. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmid, YAC, EBV derived episomes and the like. In some cases, ligation of the antibody gene into the vector provides that its transcriptional and translational regulatory sequences within the vector provide its intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to compete with the expression host cell used. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors or the same expression vector. The antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction sites on antibody gene fragments and vectors, or blunt terminal ligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete human C _H or C _L immunoglobulin sequence in which the appropriate restriction site is engineered so that any V _H or V _L sequence can be inserted and expressed as described herein. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site in front of the human C domain, and also occurs in the splice region occurring within the human C _H exon. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. Recombinant expression vectors can also encode signal peptides that facilitate secretion of the antibody chain from the host cell. The antibody chain gene can be encoded with a vector so that the signal peptide is in-frame linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vector carries regulatory sequences that regulate the expression of the antibody chain genes in the host cell. Those skilled in the art understand that the design of the expression vector, including the selection of regulatory sequences, may vary depending on factors such as the choice of host cell to be transformed, the expression level of the protein of interest, and the like. Preferred regulatory sequences for mammalian host cell expression include viral components that direct high levels of protein expression in mammalian cells, such as retrovirus LTR, cytomegalovirus (CMV) (eg, CMV promoter / enhancer), apes virus 40 (SV40). ) (Eg, SV40 promoter / enhancer), promoters derived from adenoviruses (eg, adenovirus major late promoters (AdMLP)) and / or enhancers, polyomas, and potent mammalian promoters such as native immunoglobulins and actins It includes a promoter. For further description of viral regulatory components and sequences thereof, see, eg, US Pat. No. 5,168,062, US Pat. No. 4,510,245, and US Pat. No. 4,968,615. Methods of expressing antibodies in plants, including promoters and vectors, and descriptions of plant transformation, are known in the art. See, for example, US Pat. No. 6,517,529, which is incorporated herein by reference. Methods of expressing polypeptides in bacterial cells or fungal cells, such as yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors can carry additional sequences, such as sequences that control the replication of the vector in a host cell (eg, origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vectors are introduced (see, eg, US Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, which are incorporated herein by reference). For example, typically selectable marker genes confer resistance to drugs such as G418, hygromycin or methotrexate on host cells into which the vector is introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification), the neo gene (for G418 selection) and glutamate synthetase (GS) Genes.

Non-hybridoma  Recombinant production of host cells and proteins

Nucleic acid molecules encoding antiCCR2 antibodies and vectors comprising the nucleic acid molecules can be used for the transfection of suitable mammalian, plant, bacterial, insect or yeast host cells. Transformation may be by any known method for introducing polynucleotides into host cells. Methods of introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electrophoresis, liposomes of polynucleotide (s). Encapsulation and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, US Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, which are incorporated herein by reference. For example, methods for transforming plant cells, including Agrobacterium mediated transformation, infusion transformation, direct injection, electrophoresis and viral transformation, are well known in the art. Methods of transforming bacteria, insect cells and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). This is especially true for Chinese hamster ovary (CHO) cells, NS0 cells, NSO cells, SP2 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human Hepatocellular carcinoma cells (eg Hep G2), A549 cells and numerous other cell lines. Particularly preferred cell lines are selected through determination of whether the cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When introducing a recombinant expression vector encoding an antibody gene into a mammalian host cell, the antibody is expressed by culturing the host cell for a period of time sufficient to express the antibody in the host cell or to secrete the antibody into the culture medium in which the host cell is cultured. To prepare. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, nicotiana, cephalopods, frogweed rice, corn, wheat, potatoes and the like. Bacterial host cells E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

In addition, numerous known techniques can be used to enhance the expression of antibodies from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a conventional approach to enhancing expression under certain conditions. GS systems are discussed in whole or in part in connection with EP 216 846, 256 055, 323 997 and 338 841.

Antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present disclosure regardless of the glycosylation of the antibody.

Transgenic Animals and Plants

In addition, anti-CCR2 antibodies can be produced by transformation through the production of mammals or plants that are transforming the immunoglobulin heavy and light chain sequences of interest and the production of recoverable forms of antibodies therefrom. With regard to the production of transformation in mammals, anti-CCR2 antibodies can be prepared and recovered from the milk of goats, cattle or other mammals. See, for example, US Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957, which are incorporated herein by reference. In some cases, non-human transgenic animals comprising human immunoglobulin loci are immunized with CCR2 or an immunogenic portion thereof as described herein. Methods of making antibodies in plants are described, for example, in US Pat. Nos. 6,046,037 and 5,959,177, which are incorporated herein by reference.

In some cases, one or more nucleic acid molecules encoding anti-CCR2 antibodies are introduced into an animal or plant by standard transformation techniques to produce a non-human transgenic animal or plant. See Hogan, supra, and US Pat. No. 6,417,429. The transformed cells used to prepare the transgenic animals can be embryonic stem cells or somatic cells or fertilized eggs. Transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See, eg, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual second ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999), all of which are incorporated herein by reference. In some cases, transgenic nonhuman animals have targeted cleavage and replacement by targeting the constructs encoding the heavy and / or light chains of interest. In one case, the transgenic animal specifically binds to CCR2, preferably (iii) binds to the first and / or second extracellular loop of CCR2; (Ii) does not bind to the N-terminal end or the third extracellular loop or both of CCR2; Or (iii) a nucleic acid molecule encoding both heavy and light chains. In one case, the transgenic animal comprises and expresses nucleic acid molecules encoding heavy and light chains that specifically bind human CCR2. In some cases, transgenic animals include nucleic acid molecules encoding modified antibodies, such as single chain antibodies, chimeric antibodies or humanized antibodies. Anti-CCR2 antibodies can be made in any transgenic animal. In one case, the non-human animal is a mouse, rat, sheep, pig, goat, cow or horse. Non-human transgenic animals express the encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other body fluids.

Phage  Display library

The present invention provides an anti-CCR2 antibody comprising synthesizing a library of human antibodies on phage, screening a library having CCR2 or a portion thereof, isolating phage that binds to CCR2, and obtaining an antibody from the phage; or Provided are methods for generating antigen binding portions thereof. By way of example, a method for preparing a library of antibodies for use in phage display techniques comprises immunizing a non-human animal comprising a human immunoglobulin locus with CCR2 or an antigenic portion thereof to generate an immune response, from the immunized animal Extracting antibody producing cells; Isolating RNA encoding the heavy and light chains of the antibody from the extracted cells, reverse transcription of the RNA to generate cDNA, amplifying the cDNA using a primer, and inserting the cDNA into a phage display vector to insert the antibody on phage. Expressing the same. In this way, recombinant anti-CCR2 antibodies can be obtained.

Recombinant combinatorial antibody libraries can be screened to isolate recombinant anti-CCR2 human antibodies. The library can be an scFv phage display library generated using human V _L and V _H cDNA prepared from mRNA isolated from B cells. Methods of making and screening these libraries are known in the art. Kits for generating phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, Catalog 27-9400-01; and Stratagene SurfZAP ™ Phage Display Kit, Catalog 240612). There are also other methods and reagents that can be used to generate and screen antibody display libraries (eg, US Pat. No. 5,223,409; PCT Publications WO 92/18619, WO 91/17271, WO Pat. 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio / Technology 9: 1370-1372 (1991 Hay et al., Hum.Antibod.Hybridomas 3: 81-85 (1992); Huse et al., Science 246: 1275-1281 (1989); McCafferty et al., Nature 348: 552-554 (1990) Griffiths et al., EMBO J. 12: 725-734 (1993); Hawkins et al., J. Mol. Biol. 226: 889-896 (1992); Clackson et al., Nature 352: 624-628 ( (1991); Gram et al., Proc. Natl. Acad. Sci. USA 89: 3576-3580 (1992); Garrad et al., Bio / Technology 9: 1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res. 19: 4133-4137 (1991); Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982 (1991), all of which are incorporated herein by reference).

In one case, the epitopes described in PCT Publication WO 93/06213 (incorporated herein by reference) using the human antiCCR2 antibodies described herein for the first time to isolate and produce human antiCCR2 antibodies with desired properties. Imprinting methods are used to select human heavy and light chain sequences having similar binding activity for CCR2. Antibody libraries used in this method are described in PCT Publication WO 92/01047, McCafferty et al., Nature 348: 552-554 (1990); Griffiths et al., EMBO J. 12: 725-734 (1993), all incorporated herein by reference], which may be scFv libraries prepared and screened. ScFv antibody libraries can be screened using human CCR2 as an antigen.

Once the initial human V _L and V _H domains are selected, a "mix and match" experiment is performed, where several pairs of V _L and V _H segments initially selected for CCR2 binding are screened for V _L. Select the / V _H pair combination. In addition, in order to further improve the quality of the antibody, a process similar to the in vivo somatic mutation process responsible for the affinity maturation of the antibody during the natural immune response, preferably in the CDR3 region of V _H and / or V _L is preferred. The V _L and V _H segments of the _L / V _H pair (s) can be randomly mutated. This in vitro affinity maturation can be accomplished by amplifying the V _H and V _L domains using PCR primers complementary to V _H CDR3 or V _L CDR3, respectively, which primers are capable of any of four nucleotide bases at several positions. is "interference" by the mixture, the resulting PCR product is encoding the V _H and V _L segment which is a random mutation is introduced into the V _H and / or V _L CDR3 region. These optional mutated V _H and V _L segments can be rescreened for binding to CCR2.

After screening and isolation of anti-CCR2 antibodies from recombinant immunoglobulin display libraries, nucleic acids encoding the selected antibodies can be recovered into display packages (eg from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. Can be. If desired, the nucleic acid can be further manipulated to generate other antibody forms as described herein. To express recombinant human antibodies isolated by screening of combinatorial libraries, DNA encoding the antibodies is cloned into recombinant expression vectors and introduced into mammalian host cells as described herein.

Class switching

Another aspect provides a method of converting a class or subclass of an anti-CCR2 antibody to another class or subclass. In some cases, nucleic acid molecules encoding V _L or V _H that do not include sequences encoding C _L or C _H are isolated using methods well known in the art. The nucleic acid molecule is then operably linked to a nucleotide sequence encoding C _L or C _H from the desired immunoglobulin class or subclass. This can be accomplished using vectors or nucleic acid molecules comprising a C _L or C _H chain as described herein. For example, anti-CCR2 antibodies that were originally IgM can be class switched to IgG. Class switching can also be used to convert one IgG subclass to another IgG subclass, for example from IgG1 or IgG2 to IgG4. Another method of producing an antibody comprising the desired isotype comprises isolating a nucleic acid encoding a heavy chain of an anti-CCR2 antibody and a nucleic acid encoding a light chain of an anti-CCR2 antibody, isolating a sequence encoding a V _H region, Ligating the V _H sequence to a sequence encoding a heavy chain constant domain of a desired isotype, expressing a light chain gene and heavy chain construct in a cell and collecting an antiCCR2 antibody having the desired isotype.

De-immunized  Antibodies

In another embodiment, the antibody is de-immunized to reduce its immunogenicity, for example using techniques described in PCT Publications WO 98/52976 and WO 00/34317, incorporated herein by reference. Can be.

Mutated  Antibodies

In another embodiment, nucleic acid molecules, vectors, and host cells can be used to prepare mutated antiCCR2 antibodies. Antibodies can be mutated in the variable domains of the heavy and / or light chains, for example to alter the binding properties of the antibody. For example, one or more of the CDRs can be mutated to increase or decrease the K _D of the antibody relative to CCR2, to increase or decrease k _off or to alter the binding specificity of the antibody. Techniques in site-directed mutations are well known in the art. See, eg, Sambrook et al. And Ausubel et al., Supra. In another case, one or more mutations may occur in amino acid residues known to be altered in comparison to the gonads in monoclonal antibody 4.40, 4.9, 4.22, 4.39 or 4.40 A68G S230P. Mutations may occur in the CDR or framework regions of the variable domains, or in constant domains. In one case, mutations may occur in the variable domains. In some cases, CDRs or frames of amino acid sequence variable domains selected from SEQ ID NO: 11, SEQ ID NO: 47, SEQ ID NO: 83, SEQ ID NO: 176, SEQ ID NO: 29, SEQ ID NO: 65, SEQ ID NO: 101, SEQ ID NO: 194, or SEQ ID NO: 113 One or more mutations may occur in amino acid residues known to have been altered in comparison to the gonads in the work zone.

In another embodiment, the framework region is mutated so that the resulting framework region (s) have the amino acid sequence of the corresponding germline gene. Mutations can occur in framework regions or constant domains to increase the half-life of anti-CCR2 antibodies. See, eg, PCT Publication WO 00/09560, which is incorporated herein by reference. To alter or reduce the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, to add or remove one or more glycosylation sites or to complement fixation, FcR binding and antibody dependent cell mediation Mutations in framework regions or constant domains can also occur to alter properties such as cytotoxicity (ADCC). A single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domains or in the constant domains.

In some cases, there are 1 to 8 amino acid mutations (including any number between them) in either the V _H or V _L domain of the mutated antiCCR2 antibody as compared to the antiCCR2 antibody prior to the mutation. In any of the above, mutations may occur in one or more CDRs. In addition, any mutation may be a conservative amino acid substitution. In some cases, there are no more than 5, 4, 3, 2 or 1 amino acid alterations in the constant domains.

Modified antibodies

In another embodiment, a fusion antibody or immunoconjugate comprising all or a portion of an antiCCR2 antibody linked to another polypeptide can be prepared. In one case, only the variable domain of the antiCCR2 antibody is linked to the polypeptide. In yet another case, the V _H domain of the anti-CCR2 antibody is linked to the first polypeptide and the V _L domain of the anti-CCR2 antibody is such that the V _H and V _L domains can interact with each other to form an antigen binding site. To a second polypeptide that associates with the first polypeptide. In yet another case, the V _H domain can be separated from the V _L domain by a linker such that the V _H and V _L domains interact with each other (see below under a single chain antibody). The V _H -linker-V _L antibody is then linked to the polypeptide of interest. Fusion antibodies are useful for directing polypeptides to CCR2 expressing cells or tissues. The polypeptide may be a therapeutic agent such as a toxin, chemokine or other regulatory protein or may be a diagnostic agent such as an enzyme that can be readily visualized such as mustard peroxidase. In addition, two (or more than two) single-chain antibodies can produce fusion antibodies linked to one another. This is useful when you want to generate bivalent or multivalent antibodies on a single polypeptide chain or when you want to generate bispecific antibodies.

To generate a single chain antibody (scFv), a DNA fragment encoding V _H and V _L is operative to another fragment encoding a flexible linker, eg, encoding an amino acid sequence (Gly ₄ -Ser) ₃ . Linked to, the V _H and V _L sequences can be expressed as contiguous single chain proteins while the V _L and V _H domains are linked by a flexible linker. See, eg, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); McCafferty et al., Nature 348: 552-554 (1990). Single chain antibodies may be monovalent when only single V _H and V _L are used, or bivalent if two V _H and V _L are used, or when more than two V _H and V _L are used It can be approaching. It is possible to generate bispecific or multivalent antibodies that specifically bind to CCR2 and another molecule.

In another embodiment, other modified antibodies can be prepared using anti-CCR2 antibodies encoding nucleic acid molecules. Using standard molecular biological techniques in accordance with the teachings of the specification, for example, "kappa body" (Ill et al., Protein Eng. 10: 949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13: 5303-9 (1994)), "binary antibody" (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)) or "Janusin" (Traunecker et al. EMBO J. 10: 3655-3659 (1991); Traunecker et al., Int. J. Cancer (Suppl.) 7: 51-52 (1992).

Bispecific antibodies or antigen binding fragments can be prepared by a variety of methods, including fusion of hybridomas or binding of Fab ′ fragments. See, eg, Songsivilai & Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In addition, bispecific antibodies can be formed as "biantibodies" or "Janusin". In some cases, bispecific antibodies bind to two different epitopes of CCR2. In some cases, bispecific antibodies have a first heavy and first light chain and additional antibody heavy and light chains from monoclonal antibody 4.40, 4.9 or 4.40 A68G S230P. In some cases, additional light and heavy chains are also derived from one of the monoclonal antibodies identified above, but differ from the first heavy and light chains.

In some cases, the modified antibodies described herein are prepared using one or more of the variable domains or CDRs from human anti-CCR2 monoclonal antibodies provided herein.

Induced and Labeled  Antibodies

The anti-CCR2 antibody or antigen binding moiety can be derivatized or linked to another molecule (eg another peptide or protein). Generally, the antibody or part thereof is derivatized such that CCR2 binding is not negatively affected by derivatization or labeling. Thus, antibodies and antibody portions are intended to include both intact and modified forms of the human anti-CCR2 antibodies described herein. For example, the antibody or portion of an antibody may be comprised of one or more other molecular aggregates (eg, by means of chemical coupling, genetic fusion, non-covalent association, or else), such as another antibody (eg, bispecific antibody or dual antibody), May be functionally linked to a protein or peptide (eg, streptavidin core region or polyhistidine tag) capable of mediating the association of the antibody or portion of the antibody by a detection agent, cytotoxic agent, pharmaceutical agent and / or another molecule. .

One type of derivatized antibody is prepared by crosslinking two or more antibodies (e.g., of the same or different types) to produce a bispecific antibody. Suitable crosslinkers are heterobifunctional (e.g. m-maleimidobenzoyl-N-hydroxysuccinimide esters) having two clearly reactive groups separated by a suitable spacer or homobifunctional (e.g. And disuccinimidyl suverrate). The linker is available from Pierce Chemical Company (Rockford, Illinois, USA).

Another type of derivatized antibody is a labeled antibody. Useful detection agents from which the antibody or antigen binding moiety can be derivatized include fluorescein, fluorescein isothiocyanate, rhodamine, phycoerythrin, 5-dimethylamine-1-naphthalenesulfonyl chloride, lanthanum-based phosphors And fluorescent compounds including these. Antibodies can also be labeled by enzymes useful for detection, such as, for example, mustard peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When the antibody is labeled with a detectable enzyme, it is detected by the addition of additional reagents that use enzymes that produce an identifiable reaction product. For example, when mustard peroxidase material is present, hydrogen peroxide and diaminobenzidine are added to produce a detectable colored reaction product. Antibodies can also be labeled by biotin and detected via indirect measurement of avidin or streptavidin binding. Antibodies can also be labeled by predetermined polypeptide epitopes (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) recognized by secondary reporters. In some cases, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Anti-CCR2 antibodies can also be labeled by radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect CCR2 expressing tumors by X-ray or other diagnostic techniques. In addition, radiolabels can be used therapeutically as toxins for cancerous cells or tumors. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides of ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I and ¹³¹ I. Do not.

AntiCCR2 antibodies can also be derivatized by chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. This group is useful for improving the biological characteristics of the antibody, for example to increase serum half-life or to increase tissue binding.

In some cases, anti-CCR2 antibodies can be labeled with paramagnetic, radioactive or fluorescent ions or moieties detectable upon imaging.

In some cases, paramagnetic ions are selected from chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), and samarium (III). ), Ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). In other cases, the radioactive ions are iodine 123, technetium 99, indium 111, rhenium 188, rhenium 186, copper 67, iodine 131, yttrium 90, iodine 125, astatin 211 and gallium 67. In other cases, anti-CCR2 antibodies are labeled with X-ray contrast agents such as lanthanum (III), gold (III) lead (II) and bismuth (III).

Pharmaceutical Compositions and Kits

The present invention provides compositions comprising human anti-CCR2 antibodies with antagonist properties. The composition includes liver fibrosis, kidney fibrosis, pulmonary fibrosis, psoriasis in which CCR2 plays a role; Inflammatory diseases, allergic diseases, autoimmune diseases, graft rejection, atherosclerosis, obesity, HIV infection, neuropathic pain, ischemia, stenosis and restenosis related inflammation, cancer, sepsis, scleroderma and diabetes It is useful for treating conditions that are not limited to. In some cases, treatment of liver fibrosis mediated by hepatitis C virus (HCV), hepatitis B virus (HBV), nonalcoholic fatty hepatitis (NASH) and / or alcohol-induced fatty hepatitis (ASH). In some cases, the subject needs to reduce leukocyte infiltration into tissue, such as tissue that is the site of an inflammatory response. In some cases, the subject of treatment is a human. In other cases, the subject is a veterinary subject. Examples of tissues that require reduced inflammation or reduced white blood cell infiltration include brain, spinal cord and peripheral nervous system, heart, blood vessels, esophagus, stomach, small intestine, large intestine, large intestine, prostate, pancreas, urinary tract, ovary, breast, uterus, testes, penis , Connective tissue including bones, muscles, thyroid gland, adrenal gland, pituitary gland, adipose tissue, bone marrow, blood, thymus, spleen, lymph nodes, skin, eyes, ears or nose, cartilage, liver, lung, kidney, nerve tissue However, they are not limited thereto. In one case, the tissue is a tissue with a mucosal surface.

As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are, by way of example only, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it may be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the half-life or effectiveness of the wetting agent or antibody.

The composition may be in a variety of formulations, such as liquid, semisolid and solid formulations, such as liquid solutions (eg, injectable and dropping solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The formulation depends on the intended mode of administration and therapeutic use. Conventional compositions are in the form of injectable or injectable solutions, such as compositions similar to those used for passive immunization of humans. Preferred modes of administration are parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In one case, the antibody is administered by intravenous infusion or injection. In yet another case, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of anti-CCR2 antibody in the appropriate solvent with one or a combination of ingredients enumerated above as necessary and then aseptically filtering. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required ingredients other than those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are lyophilization and vacuum drying to produce a powder of the active ingredient + any further desired ingredient from its already sterile filtered solution. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. For example, agents that delay absorption such as monostearate salts and gelatin may be included in the composition to prolong the absorption of the injectable composition.

Although antibodies can be administered by a variety of methods known in the art, for many therapeutic uses, the preferred route / administration of administration is subcutaneous, intramuscular or intravenous infusion. As will be appreciated by those skilled in the art, the route and / or mode of administration may vary depending upon the desired result. Other modes of administration include intraperitoneal, intrabronchial, transmucosal, spinal cord, intravitreal, intraaortic, intranasal, eye, ear, topical and buccal, and intratumoral.

In some cases, the active compounds of an antibody composition can be prepared by a carrier that protects the antibody against rapid release, such as controlled release formulations including implantation, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, eg, Sustained and Controlled Release Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

Additional active compounds may also be incorporated into the compositions. In some cases, the inhibitor anti-CCR2 antibody is co-formulated and / or coadministered with one or more additional therapeutic, diagnostic or prophylactic agents. Therapeutic agents include anti-CCR2 antibodies with different microspecificities, antibodies that bind to other targets, photosensitizers, androgens, estrogens, nonsteroidal anti-inflammatory agents, antihypertensives, analgesics, antidepressants, antibiotics, anticancer agents, anesthetics, anti-nausea drugs, anti-infective agents, Contraceptives, antidiabetic agents, steroids, antiallergic agents, chemotherapy agents, migraine medications, anti-smoking preparations, antiviral agents, immunosuppressants, thrombolytic agents, cholesterol lowering agents and anti-obesity agents.

Therapeutic agents also include peptide analogs that inhibit CCR2, antibodies or other molecules that bind to MCP-1, MCP-2, MCP-3 or MCP-4 and prevent its binding to CCR2 and agents that inhibit CCR2 expression. . In one case, additional agents that inhibit CCR2 expression include antisense nucleic acids that can hybridize to CCR2 mRNA, such as hairpin RNA or siRNA. Sequence specific nucleic acids capable of inhibiting gene function by RNA interference are well known in the art. Such combination therapies may require small doses of inhibitor anti-CCR2 antibodies, and agents administered in combination, thus avoiding possible toxicity or complications associated with various monotherapy.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is an antimicrobial agent. Antimicrobial agents include antibiotics (eg, antibacterial agents), antiviral agents, antifungal agents, and antiprotozoal agents. Examples of antimicrobial agents include, but are not limited to, sulfonamide, trimetaprim-sulfamethoxazole, quinolone, penicillin, and cephalosporin.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody is a chemokine antagonist, a CCR2 antagonist, an MCP-1 antagonist or a CCR5 antagonist. CCR2 antagonists or MCP-1 antagonists include antibodies associated with MCP-1 (US Pat. Nos. 7,202,343, US2005 / 0025768, US2006 / 0039913, US2006 / 0246069 and US2004 / 004/0047860); Tetrahydropyranyl cyclopentyl benzylamide compound (US2006 / 0116421); Heteroarylpiperidine compounds (US2005 / 0250781); Piperidinyl cyclopentyl aryl benzylamide compounds (US2006 / 0173013); Cyclic amine compounds (US Pat. No. 6,140,349 and US Pat. No. 6,476,054); Cyclopentyl compounds (US2002 / 0049222 and US Pat. No. 6,545,023); Tetrahydropyranyl cyclopentyl tetrahydropyridopiperidine compounds (US2004 / 0167156, US Pat. No. 6,812,234 and US Pat. No. 7,230,008); Aminocyclopentyl condensed heterotricyclicamide compounds (US2007 / 0004714); Piperidinyl-alpha-aminoamide compounds (US2005 / 0250814); Substituted pyrazole compounds (WO06 / 88813); Tetrahydropyranyl cyclopentyl heterocyclic amide compound (US2006 / 0178363); 7- and 8-membered heterocyclic cyclopentyl benzylamide compounds (US2006 / 0183731); Benzoxazinyl-amidocyclopentyl-heterocyclic compound (US2006 / 0069088); 3-aminocyclopentanecarboxamide compound (US2007 / 0149532); Nitrogen-containing heterocyclic compounds (US2007 / 0155713); Triazolyl phenyl benzenesulfonamide compound (WO 08/10934); Substituted pyrrolidine derivatives (US2004 / 0186140); Substituted benzamide and substituted phenylcarbamate derivatives (US Pat. No. 7,087,604); Substituted cycloalkylamine compounds (US2005 / 0054626); Substituted bicycloalkylamine compounds (US2005 / 0227960); Pyrrolidinone and pyrrolidine-thione compounds (US2003 / 0149081, US Pat. No. 6,727,275 and US Pat. No. 6,936,633); Mercaptoimidazole compounds (US2007 / 0244138); Substituted dipiperidine compounds (US2007 / 0197590); Phenylamino substituted quaternary salt compounds (US2006 / 0293379); Bicyclic and bridged nitrogen heterocycles (US2006 / 0074121); 3-aminocyclopentanecarboxamide compound (US2006 / 0020133); 3- (4-heteroarylcyclohexylamino) cyclopentanecarboxamide compound (US2005 / 0267146); Triazolo compounds (US Pat. No. 6,492,364); Heteroaryl sulfonamide compounds (US2006 / 0173019); Aryl sulfonamides (US Pat. No. 6,939,885); Bis-aryl sulfonamides (US Pat. Nos. 7,227,035 and US2004 / 0167113); Substituted benzamide compounds (US Pat. No. 6,821,964); Substituted diazepam compounds (US2007 / 0249589); Triazaspiro [5.5] undecane derivatives (US2005 / 267114); Substituted piperidinecarboxamide compounds (US2003 / 0114443 and US6,562,978); Benzazepine derivatives (US2004 / 0235822 and US Pat. No. 7,262,185), but are not limited to these.

If selected, the chemokine antagonist co-formulated or co-administered with the CCR2 antibody is chemically 4,4-difluoro-N-{(1S) -3- [exo-3- (3-isopropyl-5- Methyl-4H-1,2,4-triazol-4-yl) -8-azabicyclo [3.2.1] oct-8-yl] -1-phenylpropyl} cyclohexanecarboxamide, known as SELZENTRY ™ ( Maravilock):

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is a CB-1 receptor antagonist. As used herein, the term “CB-1 receptor” refers to a G-protein coupled type 1 cannabinoid receptor. The term “antagonist” includes both full and partial antagonists and inverse agonists. CB-1 receptor antagonists may be selective for CB-1 receptors. "Selective CB-1 receptor" means a compound with little or no activity antagonizing the cannabinoid-2 receptor (CB-2). CB-1 antagonists may be at least about 10-fold selective for the CB-1 receptor as compared to the CB-2 receptor. For example, the inhibitor concentration (IC50) that antagonizes the CB-1 receptor is about 10 times lower than antagonizing the CB-2 receptor. Suitable CB-1 receptor antagonists include US Pat. No. 5,462,960; 5,596,106; 5,596,106; 5,624,941; 5,624,941; 5,747,524; 5,747,524; 6,017,919; 6,017,919; 6,028,084; 6,028,084; No. 6,432,984; 6,476,060; 6,476,060; No. 6,479,479; No. 6,518,264; And 6,566,356; US Patent Publication No. 2003/0114495; US2004 / 0077650; US2004 / 0092520; US2004 / 0122074; US2004 / 0157838; US2004 / 0157839; US2004 / 0214837; US2004 / 0214838; US2004 / 0214855; US2004 / 0214856; 2004/0058820: 2004/0235926; US2004 / 0259887; US2005 / 0080087; 2005/0026983 and 2005/0101592; PCT Patent Publication No. WO 03/075660; WO 02/076949; WO 01/029007; WO 04/048317; WO 04/058145; WO 04/029204; WO 04/012671; WO 03/087037; WO 03/086288; WO 03/082191; WO 03/082190; WO 03/063781; WO 04/012671; WO 04/013120; WO 05/020988; WO 05/039550; WO 05/044785; WO 05/044822; And WO 05/049615: PCT Patent Application No. PCT / IB2004 / 004050, filed December 6, 2004; PCT / IB2004 / 004017, filed December 6, 2004; PCT / IB2004 / 004023, filed December 6, 2004; And PCT / IB2004 / 004019, filed December 6, 2004; And US Provisional Application No. 60/523937, filed November 21, 2003; 60/529908, filed December 16, 2003; 60/529909, filed Dec. 16, 2003; 60/529910, filed December 16, 2003; 60/530012, filed December 16, 2003; And compounds disclosed in US Pat. No. 60/564648, filed April 21, 2004. All of these patents and patent applications are incorporated herein by reference. Preferred CB-1 receptor antagonists for use in the method include limonabant (also known as SR141716A, available under the trade name Acomplia ™), available from Sanofi-Synthelabo or prepared as described in US Pat. No. 5,624,941; N- (piperidin-1-yl) -1- (2,4-dichlorophenyl) -5- (4-iodophenyl) -4-methyl-1H, available from Tocris ™, Ellisville, Missouri, USA -Pyrazole-3-carboxamide (AM251); [5- (4-Bromophenyl) -1- (2,4-dichloro-phenyl) -4-ethyl-N- (1-piperidinyl)-, which may be prepared as described in U.S. Patent No. 6,645,985. 1H-pyrazole-3-carboxamide] (SR147778); N- (piperidin-1-yl) -4,5-diphenyl-1-methylimidazole-2-carboxamide, N, which may be prepared as described in PCT Patent Publication No. WO 03/075660 -(Piperidin-1-yl) -4- (2,4-dichlorophenyl) -5- (4-chlorophenyl) -1-methylimidazole-2-carboxamide, N- (piperidine -1-yl) -4,5-di- (4-methylphenyl) -1-methylimidazole-2-carboxamide, N-cyclohexyl-4,5-di- (4-methylphenyl) -1- Methylimidazole-2-carboxamide, N- (cyclohexyl) -4- (2,4-dichlorophenyl) -5- (4-chlorophenyl) -1-methylimidazole-2-carboxamide And N- (phenyl) -4- (2,4-dichlorophenyl) -5- (4-chlorophenyl) -1-methylimidazole-2-carboxamide; 1- [9- (4-chloro-phenyl) -8- (2-chloro-phenyl) -9H-purin-6-yl] -4-, which may be prepared as described in US Patent Publication No. 2004/0092520. Hydrochloride, mesylate and besylate salts of ethylamino-piperidine-4-carboxylic acid; 1- [7- (2-chloro-phenyl) -8- (4-chloro-phenyl) -2-methyl-pyrazolo [1,5- which may be prepared as described in US Patent Publication No. 2004/0157839. a] [1,3,5] triazin-4-yl] -3-ethylamino-azetidine-3-carboxylic acid amide and 1- [7- (2-chloro-phenyl) -8- (4- Chloro-phenyl) -2-methyl-pyrazolo [1,5-a] [1,3,5] triazin-4-yl] -3-methylamino-azetidine-3-carboxylic acid amide; 3- (4-Chloro-phenyl) -2- (2-chloro-phenyl) -6- (2,2-difluoro-propyl)-, which may be prepared as described in US Patent Publication No. 2004/0214855. 2,4,5,6-tetrahydro-pyrazolo [3,4-c] pyridin-7-one, 2- (2-chloro-phenyl) -3- (4-ethyl-phenyl) -5- (2 , 2,2-trifluoro-ethyl) -4,5-dihydro-2H-pyrrolo [3,4-c] pyrazol-6-one and 2- (2-chloro-phenyl) -3- ( 4-isopropyl-phenyl) -5- (2,2,2-trifluoro-ethyl) -4,5-dihydro-2H-pyrrolo [3,4-c] pyrazol-6-one; 3- (4-Chloro-phenyl) -2- (2-chloro-phenyl) -7- (2,2-difluoro-propyl)-, which may be prepared as described in US Patent Publication No. 2005/0101592. 6,7-dihydro-2H, 5H-4-oxa-1,2,7-triaza-azulen-8-one; 2- (2-chloro-phenyl) -6- (2,2,2-trifluoro-ethyl) -3- (4-trifluoro, which may be prepared as described in US Patent Publication No. 2004/0214838. Methyl-phenyl) -2,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one; (S) -4-chloro-N-{[3- (4-chloro-phenyl) -4-phenyl-4,5-dihydro-, which may be prepared as described in PCT Patent Publication No. WO 02/076949. Pyrazol-1-yl] -methylamino-methylene} -benzenesulfonamide (SLV-319) and (S) -N-{[3- (4-chloro-phenyl) -4-phenyl-4,5-di Hydro-pyrazol-1-yl] -methylamino-methylene} -4-trifluoromethyl-benzenesulfonamide (SLV-326); N-piperidino-5- (4-bromophenyl) -1- (2,4-dichlorophenyl) -4-ethylpyrazole-3-carbox, which may be prepared as disclosed in U.S. Patent No. 6,432,984. mid; 1- [bis- (4-chloro-phenyl) -methyl] -3-[(3,5-difluoro-phenyl) -methanesulfonyl-methylene, which may be prepared as described in US Patent No. 6,518,264 ] -Azetidine; 2- (5- (trifluoromethyl) pyridin-2-yloxy) -N- (4- (4-chlorophenyl) -3-, which may be prepared as described in PCT Patent Publication No. WO 04/048317. (3-cyanophenyl) butan-2-yl) -2-methylpropanamide; 4-{[6-methoxy-2- (4-methoxyphenyl) -1-benzofuran-3-yl] carbonyl} benzonitrile (LY-320135, which may be prepared as described in US Patent No. 5,747,524). ); 1- [2- (2,4-dichlorophenyl) -2- (4-fluorophenyl) -benzo [1,3] dioxol-5-sulfur, which may be prepared as described in WO 04/013120. Ponyl] -piperidine; And [3-amino-5- (4-chlorophenyl) -6- (2,4-dichlorophenyl) -furo [2,3-b] pyridine-, which may be prepared as described in WO 04/012671. 2-yl] -phenyl-methanone is mentioned.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is an angiogenesis inducing factor. Angiogenesis inducing factors include basic fibroblast growth factor, acidic fibroblast growth factor, vascular endothelial growth factor, engiogenin, tumor growth factor α and β tumor necrosis factor, angiopoietin, platelet induced growth factor, placental growth factor, Hepatocyte growth factor and prolipin, but is not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody is a thrombolytic. Thrombolytic agents include urokinase plasminogen activators, urokinase, streptokinase, inhibitors of α2-plasmin inhibitors and inhibitors of plasminogen activator inhibitor-1, angiotensin converting enzyme (ACE) inhibitors, spironolactone, tissue plasmin Gen activators (tPA), inhibitors of interleukin 1 alpha-converting enzymes, antithrombin III, and the like, but are not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody is an anti-obesity agent. Anti-obesity agents include apo-B / MTP inhibitors, 11β-hydroxy steroid dehydrogenase-1 inhibitors, peptide YY _{3 -36} or analogs thereof, MCR-4 agonists, CCK-A agonists, monoamine reuptake inhibitors , Sympathetic stimulant, β ₃ adrenergic agonist, dopamine agonist, melanocyte stimulating hormone receptor analog, 5-HT2c receptor agonist, melanin enrichment hormone antagonist, leptin, leptin analog, leptin receptor agonist, galanin antagonist, lipase Inhibitors, bombesin agonists, neuropeptide-Y receptor antagonists, thyroid hormone-like agents, dehydroepiandrosterone or analogs thereof, glucocorticoid receptor antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary neurotropic factors , Human aguti related protein antagonist, ghrelin receptor antagonist, histamine 3 receptor antagonist or inverse agonist and neuromedin U number Solvent agonists, but are not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody are agents that inhibit the recruitment and / or adhesion of neutrophils and / or mononuclear cells to the site of vascular injury. Such therapeutic agents can inhibit the activity (eg, binding activity, signal transduction activity) of cell surface molecules that are mediated, for example, by cell adhesion, chemotaxis and / or homing. For example, antagonists of cell adhesion molecules (e.g., integrins (e.g., β1, β2, β3, β4, β5, β6, β7, β8 integrins), selectins (e.g., E-selectin, P-selectin, L-selectin), Cadherin (e.g., E-, P-, N-cadherin), and immunoglobulin superfamily adhesion molecules (e.g., LFA-2, LFA-3, CD44 )) And antagonists of cytokine receptors (eg, antagonists of chemokine receptor function) can be co-formulated and / or coadministered with inhibitor anti-CCR2 antibodies. In addition, agents that bind to cell adhesion molecules or ligands of cytokines or chemokines and inhibit binding of ligands to receptors expressed on neutrophils and / or mononuclear cells are also co-formulated with inhibitor anti-CCR2 antibodies and / or in combination can do.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is a cardiac therapeutic agent. Examples of therapeutic agents intended to treat heart disease include growth factors, angiogenesis inducing agents, calcium channel blockers, antihypertensives, contractile promoters, antiatheromas, anticoagulants, β-blockers, antiarrhythmic agents, cardiac glycosides, anti-inflammatory agents, Antibiotics, antiviral agents, antifungal agents, and agents that inhibit protozoal infections and antitumor agents are included, but are not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody is a calcium channel blocker. Calcium channel blockers include dihydropyridine such as nifedipine, nicardipine, nimodipine and the like; Benzothiazepines such as dilitazem; Phenylalkylamines such as verapamil; Diarylaminopropylamine ethers such as bepridil; And benzimidol-substituted tetralins such as mibepradil, but are not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or coadministered with the inhibitor anti-CCR2 antibody is an antihypertensive agent. Antihypertensive agents include diuretics such as thiazides such as hydrochlorothiazide, furosemide, spironolactone, triamterene and amylolide; Antiadrenergic agents such as clonidine, guanabenz, guanfacin, methyldopa, trimetaphan, reserpin, guanetidine, guanadrel, phentolamine, phenoxybenzamine, prazosin, terrazosin, doxazosin , Propanolol, metoprolol, nadolol, atenolol, timolol, betasolol, carteolol, pindolol, acebutolol, labetalol; Vasodilators such as hydralizin, minoxidil, diazoxide, nitroprusgit; And angiotensin converting enzyme inhibitors such as captopril, benazepril, enalapril, enalapril, posinopril, ricinopril, quinapril, ramipril; Angiotensin receptor antagonists such as losartan; And calcium channel antagonists such as nifedipine, amlodipine, felodipine XL, isadipine, nicardipine, benzothiazepine (eg diltiazem) and phenylalkylamines (eg verapamil), although However, they are not limited thereto.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is an anticoagulant. Anticoagulants include heparin, warfarin, hirudin, flea anticoagulant peptides, low molecular weight heparins, such as enoxaparin, dalteparin and ardepalin, ticlopidine, danaparoid, argatroban, apsisimab and tyropiban Although it is mentioned, it is not limited to these.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is an antiarrhythmic agent. Antiarrhythmic agents include sodium channel blockers (e.g. lidocaine, procainamide, encaine, plecanide, etc.), beta adrenergic blockers (e.g. propranolol), extenders of action potential duration (e.g. Amiodarone) and calcium channel blockers (eg, bermamil, diltiazem, nickel chloride, etc.), but are not limited to these. Combination of cardiac inhibitors (e.g. lidocaine), cardiac stimulants (e.g. isoproterenol, dopamine, norepinephrine, etc.) and multiple cardiac agents (e.g. digoxin / quinidine for treating atrial fibrillation) Is also of interest.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody is an agent to treat congestive heart failure. Examples of agents for treating congestive heart failure include cardiac glycosides, contractile promoters, extracellular loop diuretics, thiazide diuretics, potassium ion-preserved diuretics, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, nitro vasodilators, phosphodiesterase inhibitors. , Direct vasodilators, α1-adrenergic receptor antagonists, calcium channel blockers, and sympathomimetic agents.

In some specific cases, the therapeutic agent (s) co-formulated and / or co-administered with the inhibitor anti-CCR2 antibody are agents suitable for treating cardiomyopathy, including, for example, dopamine, epinephrine, norepinephrine and phenylephrine.

The present invention also provides a composition for inhibiting viral infection, in particular HIV infection, in a mammal comprising a certain amount of antibody in combination with a certain amount of antiviral agent, wherein the amount of antiCCR2 antibody and the amount of antiviral agent are combined to replicate the virus, Provided are compositions that are effective for suppressing viral infection or viral load in the human body. Many antiviral agents, including nucleoside analogs (eg, AZT, 3TC and ddl), protease inhibitors and chemokine receptor antagonists, are now known in the art and can inhibit HIV infection but not viral infection.

In another embodiment, an anti-CCR2 antibody or fragment thereof may be administered in combination with other therapeutic agents, such as anti-inflammatory drugs or molecules, to a patient suffering from an inflammatory disease such as arthritis, atherosclerosis or multiple sclerosis. In one aspect, the invention provides a method of treating an inflammatory disease in a mammal comprising administering to said mammal a therapeutically effective amount of a compound in combination with an anti-inflammatory agent. Anti-inflammatory agents include any known nonsteroidal anti-inflammatory agents such as salicylic acid derivatives (aspirin), para-aminophenol derivatives (acetaminophen), indole and indene acetic acid (indomethane), heteroaryl acetic acid (ketorolac), arylpropionic acid ( Ibuprofen), anthranilic acid (mephenamic acid), enolic acid (oxycam) and alkanones (nabumethone) and their relative glucocorticoids (metabolic) and mineralocorticoids (electrolyte regulation) activity, And any known steroidal anti-inflammatory agent that includes, but is not limited to. In another case, nonsteroidal anti-inflammatory drugs such as Aspirin (Bayer, Bufferin), Ibuprofen (Motrin, Advil), Naproxen Sodium (Aleve), Ketoprofen (Orudis KT), Indomethane (Indocin), Etodolac (Lodine), diclofenac sodium (Voltaren), ropecoxib (Vioxx), celecoxib (Celebrex), in combination with Nalamethone (Relafen) or steroids such as prednisone, prednisolone, examemethasone, beclomethasone, boo Anti-CCR2 antibody is administered in combination with desonide, fluticasone or triamcinolone.

In addition, other drugs used in the treatment of inflammation include, but are not limited to, antagonists such as all histamine and bradykinin receptor antagonists, leukotriene and prostaglandin receptor antagonists and platelet activator receptor antagonists. In yet another case, the antibody or combination therapy is administered in combination with radiation therapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy treatment, i. In yet another case, the antibody is administered in conjunction with another antibody. For example, with antibodies or other agents known to inhibit inflammation, for example with antibodies or agents that inhibit alpha-4 integrins (2004/0009169) or IL-8 receptors (US2004 / 0037830). Anti-CCR2 antibodies can be administered. Additional antibodies that can be administered in combination with anti-CCR2 antibodies are described in US Pat. No. 6,6965,50; 6,406,865; 6,406,865; No. 6,352,832; And 6,084,075, the contents of which are incorporated herein by reference.

The composition may comprise a "therapeutically effective amount" or "prophylactically effective amount" of an antibody or antigen binding moiety. By "therapeutically effective amount" is meant an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of an antibody or antibody portion may vary depending on factors such as the disease state, age, sex and weight of the subject and the ability of the antibody or antibody portion to achieve the desired response in the subject. A therapeutically effective amount is also one in which the therapeutically beneficial effect is greater than any toxic or detrimental effect of the antibody or antibody portion. A “prophylactically effective amount” means an amount effective at a dose necessary for achieving the desired prophylactic result and for a period of time. Typically, since a prophylactic dose is used in a subject prior to or early in a disease state, the prophylactically effective amount can be less than the therapeutically effective amount.

Dose regimens can be adjusted to provide the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the emergency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as single doses for the mammalian subjects to be treated, each unit of an active compound calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. Contains a predetermined amount. Instructions for unit dosage forms are inherent in the art in (a) the unique characteristics of an anti-CCR2 antibody or portion thereof and the specific therapeutic or prophylactic effects to be achieved and (b) the combination of such antibodies for the treatment of hypersensitivity in a subject. It is illustrated by one limitation and depends directly on it.

Examples of non-limiting ranges for a therapeutically or prophylactically effective amount of an antibody or antibody portion are 0.025 to 50 mg / kg, 0.1 to 50 mg / kg, 0.1 to 25, 0.1 to 10 or 0.1 to 3 mg / kg. In one case, the pH range is from about 5.0 to about 6.5, from about 1 mg / ml to about 200 mg / ml of antibody, from about 1 mmol to about 100 mmol of histidine buffer, from about 0.01 mg / ml to about 10 mg / ml Of polysorbate 80 or polysorbate 20, about 100 millimoles to about 400 millimoles of non-reducing sugars (selected but not limited to trehalose or sucrose), about 0.01 millimoles to about 1.0 millimoles of disodium EDTA 2 The antibody can be administered in the formulation as a sterile aqueous solution comprising a hydrate and optionally comprising a pharmaceutically acceptable antioxidant in addition to the chelating agent. Suitable antioxidants include, but are not limited to, methionine, sodium thiosulfate, catalase, and platinum. For example, the composition may comprise methionine at a concentration ranging from 1 mM to about 100 mM, in particular about 27 mM. In some cases, the formulation comprises 5 mg / ml of the antibody in a buffer of 20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2 mg / ml polysorbate 80. Note that the dose value may vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular subject, a particular dosage regimen should be adjusted over time according to the needs of the subject and the expert judgment of the person administering or monitoring the administration, and the dosage ranges described herein are illustrative only. And are not intended to limit the scope or practice of the claimed compositions.

Another aspect provides a kit comprising an anti-CCR2 or antigen binding moiety or a composition comprising the antibody or antigen binding fragment. The kit may comprise a diagnostic or therapeutic agent in addition to the antibody or composition. Kits include, but are not limited to, instructions and packaging materials for use in diagnostic or therapeutic methods such as ice, dry ice, STYROFOAM ™, foam, plastics, cellophane, shrink wrap, bubble wrap, cardboard, and starch peanuts. May not include). In one case, the kit comprises an antibody or composition comprising the antibody and a diagnostic agent that can be used in the methods described herein. In yet another case, the kit comprises an antibody or composition comprising the antibody and one or more therapeutic agents that can be used in the methods described herein.

The present invention provides a composition and kit for inhibiting cancer in a mammal comprising a certain amount of antibody in combination with a certain amount of chemotherapeutic agent, wherein a certain amount of compound, salt, solvate or prodrug and a certain amount of chemotherapeutic agent together grow abnormal cell Provided are compositions and kits effective for inhibiting the disease. Many chemotherapeutic agents are currently known in the art. In some cases, the chemotherapeutic agent may be a mitotic inhibitor, an alkylating agent, an antimetabolite, an intercalating antibiotic, a chemokine inhibitor, a cell cycle inhibitor, an enzyme, a topoisomerase inhibitor, a biological response modifier, an antihormone Androgen and antiangiogenic agents.

Diagnostic Use

In another aspect, the present invention provides a diagnostic method. Anti-CCR2 antibodies can be used to detect CCR2 in biological samples in vitro or in vivo. In one case, the invention provides a method of injecting an antibody into a subject, determining the CCR2 expression to which the antibody is bound in the subject by localization, and expressing the expression in the subject in a normal reference subject or a standard subject. A method of diagnosing the presence or location of CCR2 expressing cells in a subject in need of diagnosis of the presence or location of CCR2 expressing cells, comprising comparing with expression and diagnosing the presence or location of cells. Anti-CCR2 antibodies can also be used as markers for inflammation and / or for infiltration of immune cells, such as monocytes, into tissues.

Anti-CCR2 antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, flow cytometry, tissue immunohistochemistry, western blot or immunoprecipitation. Anti-CCR2 antibodies can be used to detect CCR2 from humans. In another case, anti-CCR2 antibodies can be used to detect CCR2 from cynomolgus monkeys or rhesus monkeys. In another case, anti-CCR2 antibodies can be used to detect and detect CCR2 from rodents such as mice and rats.

The present invention also provides a method for detecting CCR2 in a biological sample comprising contacting the biological sample with an anti-CCR2 antibody and detecting the binding antibody. In one case, the anti-CCR2 antibody is labeled directly with a detectable label. In another case, the anti-CCR2 antibody (first antibody) cannot be labeled, and the second antibody or other molecule capable of binding the anti-CCR2 antibody can be labeled. As is well known to those skilled in the art, a second antibody is selected that can specifically bind to a particular species and class of first antibody. For example, if the anti-CCR2 antibody is human IgG, the second antibody is anti-human-IgG. Other molecules capable of binding to antibodies include, but are not limited to, Protein A and Protein G, both of which are commercially available, for example, from Pierce Chemical Co.

Suitable labels for antibodies or secondary antibodies are disclosed above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include mustard peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase, and examples of suitable prosthetic molecule complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbeliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, monosil chloride or phycoerythrin, and examples of luminescent materials May include luminol, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

In other instances, CCR2 standards in biological samples can be assessed by competitive immunoassay using CCR2 standards and unlabeled anti-CCR2 antibodies labeled by detectable substances. In this assay, the amount of labeled CCR2 standard that combines biological samples, labeled CCR2 standards and anti-CCR2 antibodies and binds to non-labelable antibodies is determined. The amount of CCR2 in the biological sample is inversely proportional to the amount of labeled CCR2 standard that binds to the anti-CCR2 antibody.

The immunoassay disclosed above can be used for a number of purposes. For example, anti-CCR2 antibodies can be used to detect CCR2 in cultured cells. In one case, anti-CCR2 antibodies are used to determine the amount of CCR2 on the surface of cells treated with various compounds. The method can be used to identify compounds that modulate CCR2 protein levels. According to the method, one sample of cells is treated with the test compound for a period of time and another sample is left untreated. If it is desired to measure total CCR2 expression, the cells are eluted and total CCR2 expression is measured using one of the immunoassays described herein. Total CCR2 expression in the treated cells versus untreated cells is compared to determine the effect of the test compound.

Preferred immunoassays for measuring total CCR2 expression are flow cytometry or immunohistochemistry. If cell surface CCR2 expression is to be measured, the cell surface level of CCR2 is measured using one of the immunoassays described herein without eluting the cells. Preferred immunoassays for determining cell surface levels of CCR2 include labeling cell surface proteins with detectable labels such as biotin or ¹²⁵ I, immunoprecipitation of CCR2 with anti-CCR2 antibodies and detecting labeled CCR2. Steps.

Another immunoassay for determining the ubiquity of CCR2, for example cell surface levels, is by the use of immunohistochemistry. Immunoassays to detect cell surface levels of CCR2 include binding of anti-CCR2 antibodies labeled by appropriate fluorophores such as fluorescein or phycoerythrin and detection of primary antibodies using flow cytometry. In another case, the anti-CCR2 antibody cannot label and labels a second antibody or other molecule capable of binding to the anti-CCR2 antibody. Methods such as ELISA, RIA, flow cytometry, western blot, immunohistochemistry, cell surface labeling and immunoprecipitation of endogenous membrane proteins are well known in the art. See, eg, Harlow and Lane, supra. In addition, the scale of immunoassays can be increased for fast screening screening to test numerous compounds for activation or inhibition of CCR2.

Anti-CCR2 antibodies can also be used to measure levels of CCR2 in tissues or in cells derived from tissues. In some cases, the tissue is a diseased disease. In some cases, the tissue is biopsy tissue. In some cases of the method, the tissue or its biopsy tissue is excised from the patient. The tissue or biopsy tissue is then used in the immunoassay to determine, for example, overall CCR2 expression, cell surface values of CCR2 or ubiquity of CCR2 by the methods described above. The method can be used to determine if the tissue expresses high levels of CCR2, which can indicate that the tissue is targeted for treatment with anti-CCR2 antibodies.

Antibodies can also be used in vivo to identify tissues and organs that express CCR2. In some cases, anti-CCR2 antibodies are used to identify CCR2 expressing cells.

The method includes administering a detectably labeled anti-CCR2 antibody or composition comprising the same to a patient in need of such a diagnostic test and imaging the patient to determine the location of CCR2 expressing tissue. Imaging analysis is well known in the medical arts and may include, but is not limited to, X-ray analysis, magnetic resonance imaging (MRI) or computed tomography (CT). The antibody can be labeled with any agent suitable for in vivo imaging, for example, a contrast agent such as barium (which may be used for X-ray analysis) or a magnetic contrast agent such as gadolinium chelate (which may be used for MRI or CT). Other labeling materials include, but are not limited to, radioisotopes such as ⁹⁹ Tc. In another case, an anti-CCR2 antibody can be labeled and can be imaged by administering a second antibody or other molecule that is detectable and capable of binding to the anti-CCR2 antibody. In another case, biopsy tissue is obtained from the patient to determine if the tissue of interest expresses CCR2.

In some cases, detectably labeled antiCCR2 comprises a fluorophore. In some cases, the fluorophore is a near-infrared fluorescent dye, dinitrophenyl, fluorescein and derivatives thereof, rhodamine, rhodamine derivatives, phycoerythrin, phycocyanin, allophycocyanine, o-phthalaldehyde and fluoresamine , TEXAS RED ™, RHODAMINE GREEN ™, OREGON GREEN ™, CASCADE BLUE ™, Picoerythrin, CY3 ™, CY5 ™, CY2 ™, CY7 ™, Coumarin, Infrared 40, MR 200, IRD 40, ALEXA FLUOR ™, Tetramethyl Rhodamine, PACIFIC BLUE ™, SYBR ™ and BODIPY ™. In the other cases, the fluorophore is showing an emission maximum in the parentheses ㎚ CY2 ™ (506), GFP ( Red Shifted) (507), YO-PRO ® -1 (509), YOYO ® -1 (509), Calcein (517), FITC (518), FLUORX ^® (519), ALEXA ^® (520), Rhodamine 110 (520), 5-FAM (522), OREGON GREEN ^® 500 (522), OREGON GREEN ^® 488 (524) , RIBOGREEN ^® (525), RHODAMINE GREEN ^® (527), Rhodamine 123 (529), MAGNESIUM GREEN ^® (531), CALCIUM GREEN ^® (533), TO-PRO ^® -1 (533), TOTO ^® -1 (533 ), JOE (548), BODIPY ^® 530/550 (550), Dil (565), BODIPY ^® (568), BODIPY ^® 558/568 (568), BODIPY ^® 564/570 (570), CY3 ^® (570) , ALEXA ^® 546 (570), TRITC (572), MAGNESIUM ORANGE ^® (575), Phycoerythrin R & B (575), Rhodamine Phalloidin (575), CALCIUM ORANGE ^® (576), Pyronin Y (580), Rhodamine B (580) , TAMRA (582), Rhodamine RED ^® (590), CY3.5 ^® (596), ROX (608), CALCIUM CRIMSON ™ (615), ALEXA ^® 594 (615), TEXAS RED ™ (615), Nile Red ( 628), YO-PRO ^® -3 (631), YOYO ^® -3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-P One of the compounds of RO ^® -3 (660), TOTO ^® -3 (660), DiD DilC (5) (665), CY5 ™ (670), Thiadicarbocyanine (671) and Cy5.5 ™ (694). have.

Therapeutic Use

In another aspect, the present invention provides a method of inhibiting CCR2 activity by administering an anti-CCR2 antibody to a patient in need thereof. Any type of antibody described herein can be used therapeutically. In various cases, the antiCCR2 antibody is a human chimeric or humanized antibody. In some cases, the antibody or antigen binding portion thereof binds to the first and / or second extracellular loop of CCR2. The antibody or antigen binding portion thereof preferably does not bind to the third extracellular loop or the N-terminal domain of CCR2.

In yet another case, CCR2 is a human and the patient is a human patient. Alternatively, the patient may be a mammal expressing CCR2 to which the anti-CCR2 antibody cross reacts. The antibody can be administered to a non-human mammal that expresses CCR2 for which the antibody cross reacts for veterinary purposes or as an animal model of human disease. This animal model may be useful for evaluating the therapeutic efficiency of antibodies.

In one aspect, the invention provides a method of administering, assisting, preventing or assisting in the treatment of a CCR2-mediated disease in a subject by administering to the subject a therapeutically effective amount of an anti-CCR2 antibody. As used herein, the term "CCR2-mediated disease" refers to or suspects that a high level of CCR2 expression or activity in a subject with a disease is responsible for or suspects a factor contributing to the pathophysiology of the disease or to exacerbation of the disease. Diseases and other diseases. For example, by increasing levels of CCR2 on the cell surface in affected cells or tissues of a diseased subject, or in cell types that contribute to the pathology of the disease or contribute to exacerbation of the disease, such as eosinophils, monocytes or lymphocytes. The disease can be assured by an increase in CCR2 mediated activity in or by an increase in the level of CCR2 ligands such as MCP-1 at the site of inflammation. For example, anti-CCR2 antibodies can be used to detect increased CCR2 expression. Increases in CCR2 activity can be detected by increased activation of G-proteins, F-actin polymerization or chemotaxis of CCR2 expressing cells, such as chemotaxis in response to MCP-1 or other CCR2 ligands.

In one aspect, the CCR2 mediated disease is characterized by fibrosis. As used herein, the term “fibrosis” refers to a pathological condition characterized by excessive deposition and metabolism of fibrosis material (eg, extracellular matrix) in response to tissue damage. In many cases, fibrosis indicates an erroneous normal healing process (ie wound healing) due to chronic or excessive tissue effects resulting in fibroblast or astrocytic activation and proliferation and collagen accumulation. Fibrosis includes vascular diseases such as heart disease, brain disease and peripheral vascular disease, and fibrotic diseases associated with all major tissue and organ systems such as eyes, skin, kidneys, lungs, digestive tract and liver (Wynn, Nature Reviews 4: 583-594 (2004); Bataller, R and Brenner, D., J. Clin. Invest. 115: 209-218 (2005)). Other sources include chemotherapeutic drugs, radiation-induced fibrosis and damage and burns. As fibrosis encompasses a broad group of pathologies, in most cases of this symptom it is believed that the general mechanism of fibrosis tissue accumulation has many factors in common. Often, the condition is initiated in response to the influx of inflammatory cells, followed by infiltration of infiltrating cells (eg, macrophages, T cells) and resting cells in tissue (eg, stellate, myofibroblast or Cooper cells). Permanent by cytokine signaling pathways. MCP-1, for example, It appears to play an important role in some diseases (Rose CE Jr, Sung SS, Fu SM.Microcirculation 10: 273-288 (2003)), and protects CCR2 deficient mice from developing pulmonary fibrosis (Moore BB, et al. Pulmonary Fibrosis in the Absence of CCR2 Signaling.J. Immunol. 167: 4368-4377 (2001)), which suggests an important role of this receptor in the lung. Similarly, perivascular cells are an important fibrotic cell type involved in the scleroderma invention, and PDGF receptor tyrosine kinase inhibitors (RTKI) seem to slow perivascular proliferation and inhibit skin lesions in patients with this progressive disease. In the kidney, leukocyte infiltration plays an important role in mediating renal tubular interstitial inflammation and fibrosis in chronic kidney disease. Vielhauer and colleagues demonstrated that in mouse models of obstructive nephropathy, expression of CCR2 and CCR5 in the accumulation of macrophages and CD3 ⁺ lymphocytes correlates with progressive fibrosis at the site of tissue injury (Vielhauer V, et al. J. Am. Soc. Nephrol. 12: 1173-1187 (2001)).

The term "fibrosis" as used herein is also used synonymously with "fibroblast accumulation and collagen deposition." Fibroblasts are connective tissue cells distributed throughout the body in connective tissue. Fibroblasts secrete non-hard extracellular matrix comprising type I and / or type III collagen. In response to damage to tissue, nearby fibroblasts or astrocytic cells migrate to the wound, proliferate, and produce large amounts of collagen extracellular matrix. Collagen is a fibrous protein rich in glycine and proline, which are major components of the extracellular matrix and connective tissue, cartilage and bone. Collagen molecules are triple-stranded helix structures called α chains wound together in a rope-like helix. Collagen exists in several forms or types, of which the most common type I is found in the skin, tendons and bones, and type III is found in the skin, blood vessels and internal organs. Examples of fibrosis include pulmonary diseases associated with fibrosis, such as idiopathic pulmonary fibrosis, radiation-induced fibrosis, chronic obstructive pulmonary disease (COPD), scleroderma, bleomycin-induced pulmonary fibrosis, chronic asthma, silicosis, asbestos-induced pulmonary fibrosis, acute lung injury And acute shortness of breath (eg, causing bacterial pneumonia, causing trauma, causing viral pneumonia, causing respiratory, non-pulmonary sepsis and aspiration); Chronic nephropathy (kidney fibrosis) associated with injury / fibrosis, such as lupus, diabetes, scleroderma, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy, hypertension, allografts, lupus and alport syndromes; Gut fibrosis, such as scleroderma and radiation induced gut fibrosis; Liver fibrosis, such as cirrhosis of the liver, alcohol-induced liver fibrosis, nonalcoholic fatty hepatitis (NASH), biliary tract injury, primary biliary cirrhosis, infection or virus-induced liver fibrosis (eg chronic HCV infection) and autoimmune infections; Head and neck fibrosis, such as radiation induced head and neck fibrosis; Corneal scars such as LASIX ™, corneal transplantation and trabeculectomy; Hypertrophic scars and keloids such as burn induction and surgery; And other fibrosis diseases such as sarcoidosis, scleroderma, spinal cord injury / fibrosis, myelofibrosis, vascular restenosis, atherosclerosis, Wegener's granulomatosis, mixed connective tissue disease, and Peyronie's disease. Does not.

In one aspect, the CCR2 mediated disease is characterized by pathological inflammation. As used herein, the term "pathological inflammation" refers to asthma, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, rheumatoid arthritis, transplant rejection, graft-versus-host disease, multiple sclerosis (particularly to inhibit further demyelination). Sclerosis), tumors, tumor metastasis, nephritis, atopic dermatitis, psoriasis, myocardial ischemia and chronic prostatitis, obesity, metabolic syndrome, and improper and / or chronic inflammation related diseases. This inflammation is characterized by a heightened response of inflammatory cells, including infiltrating leukocytes. Over time, this pathological inflammation usually damages tissues in inappropriate areas of inflammation. Accordingly, the present invention provides a method of treating a subject with pathological inflammation comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding portion thereof that binds to and inhibits CCR2.

Antibodies or antigen-binding fragments thereof can also be used to treat diseases that show that the activation of CCR2 by binding of chemokines, including MCP-1, MCP-2, MCP-3 and MCP-4, is the cause.

Respiratory allergic diseases such as asthma, allergic rhinitis, irritable lung disease, irritable pneumonia, interstitial lung disease (ILD) (eg idiopathic pulmonary fibrosis, or rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis) , ILD associated with Sjogren's syndrome, multiple myositis or cutaneous myositis, chronic obstructive pulmonary disease, anaphylaxis or hypersensitivity reactions, drug allergies (eg to penicillin, cephalosporins), bee venom allergy, inflammatory bowel disease such as Crohn's disease and Inflammatory or allergic including ulcerative colitis, spondyloarthropathy, scleroderma, psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, vasculitis (e.g., necrotic, skin and hypersensitivity vasculitis) Antagonist anti-CCR2 antibodies or antigen-binding fragments thereof for treating diseases and conditions (e.g. It can be used to 4.40 and / or 4.9 or an antigen-binding fragment).

Antagonist anti-CCR2 antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or antigen binding fragments thereof) can be used to treat autoimmune diseases. Examples of autoimmune diseases include Edison disease, hemolytic anemia, antiphospholipid antibody syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmitis, bullous analogy Celtic swelling, Celtic swelling, multiple endocrine disorders, purpura, lighter disease, Stiff-Man Syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Galantle's syndrome, insulin dependent diabetes and autoimmune inflammatory eye disease Although it is mentioned, it is not limited to these.

In some cases, the autoimmune disease treated by the antagonist anti-CCR2 antibody is rheumatoid arthritis (RA). TH1 disease, RA, is a common human autoimmune disease with a prevalence of about 1% in Caucasians (Harris, B. J. et al., 1997, In Textbook of Rheumatology 898-932), currently affecting 2.5 million Americans. RA is characterized by chronic inflammation and infiltration by activated T cells, macrophages and plasma cells of the lubricating joint, which gradually destroy artificial cartilage. This is the most common form of joint disease.

In yet another embodiment, the autoimmune disease treated by the antagonist antiCCR2 antibody is multiple sclerosis (MS). MS, also a TH1 disease, is the most common central nervous system (CNS) demyelinating disease affecting 350,000 (0.1%) individuals in North America and 1.1 million individuals worldwide. In general, MS is an autoimmune disease mediated in part by monocytes and proinflammatory CD4 T (Th1) cells that recognize specific myelin polypeptides with respect to MHC class II molecules expressed on antigen (Ag) presenting cells (APCs). It is thought to be.

Antagonist anti-CCR2 antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or 4) for treating human type I or insulin dependent diabetes mellitus (IDDM), a disease characterized by autoimmune destruction of β cells in the Langerhans pancreatic islet Antigen binding fragments thereof) can be used. Depletion of β cells makes the blood glucose levels unregulated. In humans, long pre- onset periods precede diabetes. During this period, pancreatic β cell function is progressively lost. Disease onset shows that the presence of autoantibodies to insulin, glutamic acid decarboxylase and tyrosine phosphatase IA2 (IA2).

Antagonist anti-CCR2 antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or antigen binding fragments thereof) can also be used to treat neuropathic pain. Mice deficient in CCR2 have been found to reduce neuropathic pain (Abbadie et al., Proc Natl Acad Sci U S A. 100 (13): 7947-52 (2003)). As used herein, the term "neuropathic pain" means pain resulting from damage to nerves. Neuropathic pain is distinguished from invasive pain, which is pain caused by acute tissue damage, including small skin nerves or small nerves in muscle or connective tissue. Pain, including invasive mechanisms, is usually limited to periods of tissue healing and is generally alleviated by analgesics or opioids available as described by Myers, Regional Anesthesia 20: 173-184 (1995). Neuropathic pain is usually long lasting or chronic and usually develops days or months after initial acute tissue injury. Neuropathic pain can include allodynia and persistent spontaneous pain that typically exhibit a painful response to a painless stimulus. Neuropathic pain can also be characterized by hyperalgesia, which usually exhibits a stressed response to insignificant, such as pinching, painful stimuli.

Antagonist Anti-CCR2 antibodies are useful for alleviating neuropathic pain resulting from diseases of the peripheral nerves, dorsal ganglia, spinal cord, brainstem, thalamus or cortex. The method is useful for alleviating neuropathic pain regardless of the etiology of pain. For example, peripheral neurological diseases such as neuroma; Nerve compression; Nerve crush, nerve extension or incomplete nerve cutting; The method can be used to alleviate neuropathic pain resulting from single neuropathy or multiple neuropathy. Dorsal muscle ganglia compression; Inflammation of the spinal cord; Upper left, tumor or lateral cutting of the spinal cord; Brain stem, thalamic or cortical tumors; Or the method can be used to relieve neuropathic pain resulting from diseases such as brain stem, thalamus or trauma to the cortex.

Antagonist anti-CCR2 antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or functional fragments thereof) can also be used to treat atherosclerosis. Atherosclerotic plaques develop for decades and involve inflammatory cell infiltration, smooth muscle cell proliferation, accumulation of extracellular matrix, fibrotic cap formation and angiogenesis (Bayes-Genis et al. Circ. Res. 86: 125- 130 (2000)). Chemotaxis is associated with the early onset of atherosclerosis. The cell population migrates to the inner part of the blood vessel wall, creating an endocardium, forming atherosclerotic plaques. For example, monocyte chemotaxis is caused by monocyte chemotactic protein 1 (MCP-1), which appears early in the development of atherosclerosis in the damaged arterial canal (Furukawa et al. Circ.Res. 84: 306-314 (1999). Han et al. J. Lipid Res. 40: 1053 (1999). Bone marrow transplantation from CCR2-/-mice, not CCR2 + / + mice, to ApoE3-Leiden mice (mouse strains susceptible to dietary-induced atherosclerosis) to reduce atherosclerosis, indicating that MCP-1 signaling through CCR2 causes atherosclerosis It contributes to atherosclerosis (Guo et al. (2003) Arterioscler Thromb Vasc Biol .; 23 (3): 447-53). Thus, subjects will be administered CCR2 antagonist antibodies, particularly antagonist antibodies that bind to the first and / or second extracellular loop of CCR2, to reduce the incidence of, or treat, or assist in the treatment of atherosclerosis. Can be.

Antagonist anti-CCR2 antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or antigen binding fragments thereof) can also be used to treat obesity. In obesity, adipose tissue has been shown to contain numerous monocytes. These monocytes can contribute to the development of various sequelae or fat deposits commonly associated with obesity, commonly referred to as metabolic syndrome. Metabolic syndrome includes such modifications as the development of diabetes.

Antagonist anti-CCR2 for the treatment of vasculature, especially arteries such as stenosis or restenosis of coronary arteries, such as stenosis or restenosis resulting from vascular intervention (eg surgery, therapeutic or mechanical intervention) and endometrial hyperplasia Antibodies or antigen binding fragments thereof (eg, 4.40 and / or 4.9 or antigen binding fragments thereof) may also be used. For example, restenosis, which typically causes narrowing of the luminal fissures of blood vessels, may include artificial vascular procedures, angioplasty (e.g., angioplasty performed by a balloon), atherosclerosis, laser or other suitable method (e.g., , Percutaneous coronary balloon angioplasty (PTCA), stent installation (eg, mechanical or biological endovascular stent installation), vascular bypass procedure, or a combination thereof and other procedures used to treat stenosis or occluded vessels ( But not limited to these).

Antagonist anti-CCR2 antibodies or antigen-binding fragments thereof (eg, 4.40 and / or 4.9 or the like) to treat graft rejection (eg, at the time of transplantation), including allograft rejection or graft-versus-host disease and organ transplant related arteriosclerosis. Antigen binding fragments thereof) can also be used.

As therapeutic agents for HIV infection, antibodies that are antagonists of CCR2 and antigen binding fragments thereof can be used. HIV-1 and HIV-2 are causative factors of acquired immunodeficiency syndrome (AIDS) in humans. AIDS results, in part, from a deficiency of CD4 ⁺ T lymphocytes in HIV infected subjects. HIV-1 mainly infects T lymphocytes, monocytes / macrophages, dendritic cells and, in the central nervous system, microglia. These cells both express CD4 glycoproteins that act as receptors for HIV-1 and HIV-2. Effective entry of HIV into target cells depends on the binding of the viral external envelope glycoprotein, gp120, to the amino terminal CD4 domain.

In addition, CCR2 appears to act as a cofactor for HIV-1 (Frade et al. (1997) J Clin Invest .; 100 (3): 497-502). After viral binding, the HIV-1 envelope glycoprotein mediates the fusion of viral and host cell membranes to complete the entry process. Membrane fusion, indicated by HIV-1 envelope glycoprotein expressed on infected cell surfaces, results in cell-cell fusion resulting in fusion cells.

As therapeutic agents for the treatment of various ocular conditions including aging macular degeneration (AMD), uveitis and corneal infection, antibodies and antigen binding fragments thereof which are antagonists of CCR2 can be used.

Aging  Macular degeneration ( AMD )

CCR2 appears to play an important role in the recruitment of macrophages in patients with aging macular degeneration and in the development of choroidal neovascularization (CNV) observed in patients with angioplasty, high myopia, and ocular histoplasmoma (Tsutsumi, C et al. Journal of Leukocyte Biology 74: 25-32, 2003).

Uveitis

The association between the single nucleotide polymorphism of CCR2 and its ligand (MCP-1) is characterized by acute idiopathic all uveitis (Yeo, TK, et al. Cytokine 35: 29-35 2006) and idiopathic immune mediated posterior segment uveitis (Ahad, MA, et al. Mol Vis 13: 388-396 2007).

Corneal Infection (bacteria)

CCL2 appears to play an important role in the regulation of polymorphonuclear neutrophil (PMN) recruitment during corneal infection (Xue, M. L, et al. Immunology and Cell Biology 85: 525-531 2007). Because PMN is essential for removing bacteria and promoting wound healing in the cornea, chronic inflammatory disease can develop if these cells persist.

Treatment of various solid tumors and cancers including chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, malignant myeloma, Hodgkin's disease, and carcinoma of the prostate, bladder, breast, cervix, colon, lung, liver, or stomach solid tumors and cancer As a therapeutic agent for, it is possible to use antibodies which are antagonists of CCR2 and antigen binding fragments thereof.

Prostate cancer

MCP-1 appears to act as a peripheral and autosecretory factor for prostate cancer growth and invasion (Lu, Y. et al. The Prostate 66: 1311-1318, 2006) and CCR2 expression correlates with prostate cancer progression (Lu, Y. et al. Journal of cellular biochemistry 101: 676-685, 2007). Systemic delivery of neutralizing anti-MCP-1 antibodies appears to induce prostate cancer tumor regression in mice (Loberg, R. D., Cancer Res 67: 9417-9424 2007).

Breast cancer

Tumor-associated macrophages are believed to play an important role in tumor immune surveillance and development, and activation and recruitment of lymphocytes is regulated by chemokines, including MCP-1. Significant association has been demonstrated for CCR2 polymorphisms in breast cancer (Zafiropoulos, A., N. et al. Journal of medical genetics 41: e59 2009). The CCL2 / CCR2 pathway also appears to play a central role for cancers including breast cancer, ovarian cancer and gastric cancer of myeloid suppressor cells that promote tumor regression, angiogenesis and neovascularization (Huang, B., et al. Cancer letters 252: 86-92, 2007).

Melanoma

Blocking MCP-1 function also appears to inhibit the recruitment of tumor-associated macrophages in mice and prevent tumor angiogenesis and growth of malignant melanoma (Koga, M. et al. Biochemical and biophysical research communications 365: 279-). 284, 2008).

Liver cancer

Blockade of CCR2 appears to reduce the transport of hepatic stellate cells, a major source of matrix metalloproteinase 2 that promotes neovascularization during liver tumor formation (Yang, X. et al. International journal of cancer 118: 335-). 345, 2006).

Cervical cancer

CCR2 appears to play an important role in macrophage mobilization that causes tumor angiogenesis in the development of cervical neoplasia from squamous epithelial lesions (Coelho, A., et al. Gynecologic oncology 96: 760-764, 2005; .Coelho , A., et al. Gynecologic and obstetric investigation 64: 208-212, 2007).

Ovarian Cancer

The CCL2 / CCR2 pathway also appears to play a central role in the recruitment of myeloid suppressor cells to cancer, including breast cancer, ovarian cancer and gastric cancer, which promote tumor regression, angiogenesis and neovascularization (Huang, B., et. al. Cancer letters 252: 86-92, 2007).

The antibody may be administered once or several times. Antibodies can be administered once or more every four to six months per day. Dosing three times a day, twice a day, once a day, once a day, once every three days, once a week, once a two weeks, once a month, twice a month, three months 1 It can be based on the same schedule as once a week and once every six months. The antibody can also be administered continuously via a minipump. Antibodies can be administered via mucosal, buccal, intranasal, inhaled, intravenous, subcutaneous, intramuscular, parenteral, intratumoral or topical routes. Antibodies can be administered locally or systemically.

Antibodies can be administered once, two or more times, or at least until the condition is treated, alleviated, or cured. The antibody can generally be administered as part of a pharmaceutical composition as described herein. Doses of the antibody may generally range from 0.1-100 mg / kg, 0.5-50 mg / kg, 1-20 mg / kg or 1-10 mg / kg. The serum concentration of the antibody can be measured by any method known in the art.

The following examples are for illustration only and are not to be construed as limiting the scope in any way.

Example  One

term CCR2  To produce antibodies Hybridoma  produce

8 weeks to 10 weeks old XENOMOUSE ™ mice producing human IgG2 and IgG4 antibodies at jokcheok the back thereof CCR2B (Genbank MN000648, SEQ ID NO: 204) (TITERMAX ™ Gold Adjuvant ( Sigma, catalog T2684 number, lot K1599 No.) 10 ⁷ Cells / dose / mouse; 50/50 volume preparation) and immunized with 300-19 cells transfected. Mice were treated with Aluminum Phosphate Gel Adjuvant (Catalog 1452-250, Batch 8937, HCI Biosector (5 μl / mouse / boost)) and qCpG (IMMUNEASY ™ Mouse Adjuvant) (Catalog 303101; Lot 11551249) for a period of 3 to 8 weeks. 5 to 9 additional injections in Qiagen (15 μl / mouse / boost). Four days prior to injection, mice received a final injection in PBS. Spleen and lymph node lymphocytes from immunized mice were collected and fused with nonsecretory myeloma P3-X63-Ag8.653 cell line. The fused cells were subjected to HAT selection as described above (Galfre and Milstein, Methods Enzymol. 73: 3-46 (1981)). A series of hybridomas, all secreting CCR2 specific human antibodies, were recovered. Numerous antibodies have been identified for binding to CCR2 as assessed by FACS analysis. Hybridomas were selected for further study, some of which are listed in Table 6.

The hybridomas listed in Table 2 were deposited with the American Standard Strain Conservation Authority (ATCC) (Manassas University Boulevard 10801, Virginia, USA) under the provisions of the Budapest Treaty. The following accession numbers have been assigned to hybridomas:

TABLE 2

Example  2

term CCR2  Sequence Analysis of Antibodies

To analyze the structure of the produced antibody, nucleic acids encoding heavy and light chain variable domains including fragments obtained from hybridomas producing anti-CCR2 monoclonal antibodies were cloned. The light and light chains of the CCR2 antibody were cloned and the sequence was identified as exemplified for 4.40.2 antibody as follows: Isolation of Poly (A) ⁺ mRNA using RNEASY ™ MINI KIT (Qiagen), oligo (dT) CDNA was synthesized from mRNA by ADVANTAGE RT-for-PCR kit (BD Biosciences) using priming. The primers described in Table 3 were used to amplify the oligo (dT) primed cDNA against clone 4.40.2. Amplification was performed using PFUULTRA HIGH-FIDELITY Polymerase (Stratagene) and PTC-200 DNA Engine (MJ Research) according to the following cycle: 2 min at 95 ° C .; 25X (20 seconds at 95 ° C, 30 seconds at 55 ° C, 30 seconds at 72 ° C); 10 minutes at 72 ° C. PCR amplicons were cloned into heavy and light chain expression vectors. The vector was then transformed into MAX EFFICIENCY DH5α chemically competitive cells (Invitrogen) using standard protocols. Clones were sequences identified using Grills 16 ^th BDTv3.1 / dGTP chemistry (Applied Biosystems Inc) and 3730xl DNA Analyzer (Applied Biosystems Inc). From the nucleotide sequence and the expected amino acid sequence of the antibody, gene utilization was confirmed for each antibody chain. MACVECTOR ™ and GENEWORKS ™ software programs (“V Sequence Directory” using the Oxford Molecular Group (Campbell, CA)) (MRC Center for Protein Engineering (Cambridge, UK); Tomlinson, et al., J. Mol. Biol. , 227, 776-798 (1992); Hum.Mol. Genet., 3, 853-860 (1994); EMBO J., 14, 4628-4638 (1995)).

TABLE 3 Variable domain primers designed to amplify 4.40.2 (5 '→ 3')

Table 4 lists the gene utilization of the selected hybridoma antibody clones.

[Table 4] Use of heavy and light chain genes

Hybridoma clones were generated in IgG4 mice and once the sequences were obtained, the variable domains were cloned into IgG1, IgG2 and IgG4 format expression vectors for comparison.

Example 3

Mutations of Anti-CCR2 Antibodies

Light chain  Regression from the variable region to the gonad sequence

The alanine residue at position 68 in the light chain of CCR2 4.40.2 was regressed to the germline residue glycine by site-directed mutation as follows. The primers in Table 5 were used to alter point mutations in codons for amino acid residue 68. 2 minutes at 96 ° C; 16X (50 seconds at 96 ° C, 10 seconds at 68 ° C); Site directed mutations were performed using the QUIKCHANGE ™ II site directed mutation kit (Stratagene) by standard protocols involving a 10 minute cycle modification at 68 ° C. The mutated vector was transformed into XL1-Blue supercompetent cells. To confirm the mutation was successful, clones were sequenced using Grills 16 ^th BDTv3.1 / dGTP chemistry (Applied Biosystems Inc.) and 3730xl DNA Analyzer (Applied Biosystems Inc.).

TABLE 5 Mutant primers (5 '→ 3')

The resulting antibody was termed 4.40 A68G (SEQ ID NO: 112) with glycine at position 68 of the light chain variable region.

Hinge Having a stabilizing mutation IgG4 Heavy chain  Creation of constant zones

IgG4 heavy chain constant region was isolated from pCON-G4 (pro) (Lonza (Switzerland Basel)) derived IgG4 expression vector, which was associated with IgG4 L309 allotype (Brusco A. et al., Eur J. Immunogenticsl 25 : 349-355 (1998)) amino acid 230 (Angal, S. et al. Molecular Innunology 30: 105-108 (1993)) has a hinge region stabilizing mutation (serine to proline) from the germline.

Silencing mutations (Lys, AA G → AA A ) were introduced into the 11th nucleotide residue of the G4 CH1 exon of the pCON-G4 (pro) vector by PCR mutation, removing the Apal recognition site and sub-using the following amplification primers. Promoted cloning:

(Uppercase base hybridizes (mutations of residue 11 of CH1 are bold); lowercase bases do not hybridize)

(Uppercase bases hybridize. Lowercase bases with Xbal sites do not hybridize.)

Amplification was achieved using Expand Hi-Fi polymerase (Roche) and PTC-200 DNA ENGINE ™ (MJ Research) by the following thermal cycle: 3 min at 95 ° C .; 22X (20 sec at 95 ° C., 30 sec at 58 ° C., 2 min 10 sec at 72 ° C.); 10 minutes at 72 ° C.

Another silent mutant (Thr, AC C → AC A ) was added to the G4 CH1 exon of the pCON-G4 (pro) vector to facilitate the use of an enzyme to subclone the variable region into the expression vector using the following primers. The BstEII site was removed by introduction by PCR mutation at burn nucleotide residues:

Amplification was achieved using the QUIKCHANGE ™ site directed mutation kit (Stratagene) and PTC-200 DNA Engine (MJ Research) by the following heat cycle: 1 min at 96 ° C .; 14X (50 sec at 96 ° C., 15 min 48 sec at 68 ° C.); 10 minutes at 72 ° C.

The resulting IgG4 constant region, called S230P, which includes a hinge region mutation and two silent mutations, was then subcloned into the Apal / Xbal region of the DHFR antibody expression vector as an Apal / Xbal fragment. The heavy chain variable region of the 4.40.2 antibody was then cloned into an expression vector comprising the G4 S230P constant region, resulting in a full length heavy chain construct. The 4.40 derived antibody with light chain variable region A68G germline substitutions and heavy chain hinge region S230P substitutions was named 4.40 A68G S230P having the light chain amino acid sequence of SEQ ID NO: 112 and the heavy chain amino acid sequence of SEQ ID NO: 116.

Supernatants from cells transfected with the expression vector were collected and purified by standard Protein A affinity chromatography to isolate recombinant immunoglobulins. This protein was then characterized by SDS-PAGE, light scattering and spectrometry.

Example 4

In vitro binding

1 shows in vitro binding of AF-488 conjugated antibody 4.40 A68G S230P to CCR2 transfected 300-19 cells (FIG. 1B) against human monocytes (FIG. 1A) in whole blood, and detected by anti-human PE Showing binding of various concentrations of 4.40 A68G S230P antibody to CCR2 transfected 300-19 cells (FIG. 1C). Briefly, one million cells in a volume of 100 μl were stained with test antibody using Dulbecco's phosphate buffered saline containing 2% heat inactivated fetal bovine serum and 0.002% sodium azide. After 15 minutes, the secondary detection antibody was added. After 30 minutes, cells were washed in buffer, resuspended in 500 μl and assessed for staining on FACS CALIBUR. A total of 10,000 events per tube were evaluated. Average channel fluorescence intensity was assessed for each concentration of antibody as an indicator for the scale of staining.

Example  5

term CCR2  Affinity constants of monoclonal antibodies ( K _D Determination of

To assess the K _D of the antibody against CCR2, antibody binding to 300-19 cells expressing CCR2 was evaluated by FACS in a 3 hour assay. In brief, using a buffer of Dulbecco's phosphate buffered saline containing 2% heat inactivated fetal bovine serum, 0.002% sodium azide and 0.005 mg / ml cytokalcin-B (Sigma 6762), in a volume of 100 μl One million cells were stained with test antibodies. After 15 minutes, a secondary detection antibody, R-Phycoerythrin conjugated Affini-pure F (ab ') fragment donkey antihuman IgG H + L (Jackson 709-116-149) was added. The tube was shaken gently for 3 hours at room temperature. After washing twice in buffer, cells were resuspended in 500 μl of buffer and evaluated on FACS CALIBUR ™. A total of 10,000 events per tube were evaluated. Average channel fluorescence intensity was assessed for each concentration of antibody as an indicator for the scale of staining. This binding study shows that the 4.40 A68G S230P antibody binds human CCR2 (0.58 nM) on cells transfected with 0.085 μg / mL K _D (FIG. 2). When assessing saturation binding on cells expressing CCR2 / CCR5 chimeric receptors with first and second extracellular loops of CCR2 (see Example 7), similar concentration curves and K _D (K _D = 0.023 μg / mL (0.16) nM)) was obtained as shown in FIG. 6C.

Example 6

MCP-1 Induced Chemotaxis Suppression

Human anti-CCR2 antibodies were evaluated for their ability to inhibit chemotaxis of THP-1 monocytes (ATCC number TIB 202) in response to MCP-1 (CCL2), a CCR2 ligand. As previously described (see Gladeue RP et al., J Biol Chem 278: 40473-40480 (2003)), the state in a 96-well chemotaxis chamber purchased from NeuroProbe, Inc. (Gaydersburg, MD, USA). Mars was performed. In brief, CCL2 was diluted in RPMI 1640 medium (Roswell Park Memorial Institute) containing 0.1% BSA and then added to the bottom well of the chamber. A filter (neuprobe) with a 5 μm aperture was placed between the upper well and the lower well of the chamber. THP-1 cells were then added to the top chamber (8 × 10 ⁵ ) in the presence or absence of varying concentrations of test antibodies. The instrument was incubated for 3 hours at 37 ° C. in a 5% CO ₂ wet incubator. After the incubation period, the non-migrating cells were removed from the upper chamber and the filter top was wiped. 2 mM cold EDTA was then added to the upper wells and the chemotaxis chamber was incubated at 4 ° C. for 20 minutes. EDTA was then removed and the 96 well microtiter plates were centrifuged at 800 × g for 10 minutes. The filter was then removed, the culture medium discarded, and 0.2% FDA added to the plate. The plates were then incubated for 1.5 hours at 37 ° C. until yellow. The color intensity was read on a microtiter plate reader at 490 nm to quantify the number of migrated cells. As shown in Table 6, several antibodies were identified that inhibited THP1 chemotaxis to some extent.

TABLE 6 MCP-1 Induced THP-1 Chemotaxis Inhibition

Similarly, antibody 4.40 A68G S230P inhibited chemotaxis of THP-1 monocytes in response to MCP-1 (IC 50 = 0.148 μg / ml) rather than CCR1 / CCR5 ligand MIP-1 as shown in FIG. 3.

Primary monocyte chemotaxis

Heparinized human whole blood was layered and spun down onto an ACCUSPIN HISTOPAQUE 1077 tube (Sigma, St. Louis, MO). Monocyte cell portions were collected, washed three times with PBS, erythrocyte cells (RBCs) eluted with water, and the cells were RPMI (Gibco; New York, USA) with 0.1% BSA (Sigma) and 10 mM HEPES (Gibco). Resuspended at 4 × 10 ⁶ / ml). Antibody dilutions or KLH controls were added to 0.25 nM MCP-1 (Peprotech; Rocky Hill, NJ) and 30 μl placed at the bottom of a 48 well Boyden chamber (Neuprobe; Gaithersburg, MD); The negative control was only embryo. A 5 μm PVP free filter (neuprobe) was placed thereon and the chamber sealed. Mononuclear cells isolated by antibody dilution or KLH control for 30 minutes at room temperature were incubated. 50 μl was then added to the upper wells. The chamber was incubated for 90 minutes at 37 ° C. in a 5% CO ₂ wet incubator. After incubation, cells were aspirated out of the upper wells, the filter top was wiped, air dried, stained with DIFF-QUIK ™ (Dade-Behring, Newark, Delaware, USA) and the number of cells migrated within 6 fields. Was counted. Chemotaxis inhibition of primary isolated human monocytes in response to MCP-1 by antibody 4.40 A68G S230P is shown in FIG. 4.

In addition, inhibition of THP-1 cells was not observed in the CCR1 / CCR5 ligand MIP-1a, as exemplified by the 4.40 antibody (FIG. 3).

Example  7

CCR2 Of CCR1 chimera Construction

Antibodies were tested to inhibit MCP-1 induced chemotaxis for binding to CCR2 chimeras to map epitopes. Binding of antibodies to 300-19 cells expressing multiple receptor chimeras consisting of (first, second and / or third) extracellular loops and N-terminal substitution of CCR2 by a portion of CCR1 (M = CCR2, R = This was done by evaluating CCR1).

chimera  Receptor MRRR of Construction

Using the specified QUIKCHANGE ^® region obtained from the Stratagene mutagenesis kit and the following mutagenesis primer wild type to construct the (first, second, and third extracellular loops of CCR2 (M) N-terminal, CCR1 (R) of a) MRRR chimeric receptors The first ApaL restriction enzyme site was mutated in human CCR1 and cloned into the BamHI site via the expression vector pcDNA3 (Invitrogen; Carlsbad, Calif.) Using the following primers:

FLAG tagged MMRR chimera (N terminus and first extracellular loop of CCR2, second and third extracellular loops of CCR1) cloned with pCMV-1 induced expression vector (Pharmacia; Piscataway, NJ) The chimeric receptor was provided by Israel Charo of J. David Gladstone Institutes. The construction of the MMRR chimera (called "2211" by the author) was described by Monteclaro F. S. et al. (Methods in Enzymology 228: 70-84 (1997)). In the chimera, the first ApaL site was mutated to the BamH1 site using the following mutant primers:

Each obtained mutated clone was cleaved by NdeI and BamHI. A 632 base pair NdeI / BamHI fragment containing only the CCR2 N terminus was isolated from the fragment containing the vector obtained from the CCR1 / pcDNA3 BamHI mutant and the mutated MMRR / pCMV1 plasmid, and ligated together to generate MRRR / pcDNA3 chimera.

The BamHI site in the MRRR chimera was mutated back to the ApaL site using the following mutant primers:

Complete MRRR fragments were PCR amplified using sense primer 66C (SEQ ID NO: 138) homologous to the FLAG tag region of the vector and antisense primer 66D (SEQ ID NO: 139) homologous to the 3 'end of CCR1 and comprising the HindIII site.

This fragment was then subcloned into the PflMI and HindIII sites of the pMIG (Van Parijs, L. et al., Immunity 11: 281-188 (1999)) induced retroviral constructs (FIG. 5), followed by a FLAG tag The inserted gene (mouse / human chimera) preceding the Sal1 site was replaced and the internal ribosomal entry site enhanced green fluorescent protein site (IRESEGFP) was removed.

chimera  Receptor RRRM  And RMMR of Construction

RRRM (N terminus of CCR1, first and second extracellular loop and third extracellular loop CCR2) and RMMR (N terminus and third extracellular loop of CCR1 and second and third of CCR2 using two-step PCR method 3 extracellular loop) chimeric receptors were prepared.

For RRRM chimeras, the third loop of human wild type CCR2 was initially PCR amplified using the following primers:

Wild-type human CCR2 was cloned into the retrovirus induced expression vector pMIG as a template. The N terminal region through the second extracellular loop was PCR amplified using the following primers and cloned into wild-type CCR1 as expression template pcDNA3:

These fragments were gel purified, combined and PCR amplified together using 5 'and 3' terminal primers: 89C (SEQ ID NO: 142) and 81B (SEQ ID NO: 141). The resulting fragments were ligated with pMIG containing the SalI / HindII site, N-terminal FLAG tag of the retroviral vector and with IRESEGFP removed.

chimera  Receptor RMMR of Construction

The CCR1 third extracellular loop through the cytoplasmic tail was PCR amplified using the following primers that hybridize to the 3 'region of the insert's vector in the wild type CCR1 and wild type CCR1 / pcDNA3 plasmid cloned into the expression vector pcDNA3 as a template:

PCR amplification of a second fragment comprising a region comprising the N terminus of CCR1 and the first and second extracellular loops of CCR2 using a FLAG tagged RMMM / pcDNA3 plasmid obtained from Israel Charo of J. David Gladstone Institutes as a template I was. The construction of the MMRR chimera (called "2211" by the author) was described by Monteclaro F. S. et al. (Methods in Enzymology 228: 70-84 (1997)). Primers used are sense primer 66C (SEQ ID NO: 138) and antisense chimeric primer 85B which hybridize to a FLAG tag:

These two fragments were gel purified, combined and PCR amplified together using 5 'and 3' terminal primers: 66C (SEQ ID NO: 138) and 78D (SEQ ID NO: 144). This final fragment was ligated with pflMI and HindIII and ligated into a pMIG retroviral vector containing an N-terminal FLAG tag and with IRESEGFP removed.

chimera  Expression

Retroviruses were prepared from each chimeric construct and used to transduce 300-19 cells. Expression was determined by FACS analysis using anti-FLAG epitope antibody M1 (Sigma, St. Louis, Missouri, Catalog F3040).

Example 8

Epitope mapping

FACS analysis was used to assess the binding of the antibody to the CCR2 / CCR1 chimeric receptor. All receptors were tagged at the N terminus with FLAG and also stained with anti-FLAG antibody M1 to confirm receptor expression. 6A and 6B show FACS staining of FLAG on the N terminus with M1 antibody (FIG. 6A) compared to the staining of 4.40 (FIG. 6B). 6C depicts the saturation binding curve of 4.40 A68G S230P antibody on RMMR chimeric transfected 300-19 cells. Saturated binding of 4.40 A68G S230P antibody to 300-19 cells not transfected with CCR2 showed no binding at concentrations below 10 μg / mL (FIG. 7A), but 300-19 cells transfected with fully human CCR2 Dose dependent binding is shown at concentrations of 0.01 μg / mL or higher (FIG. 7B). In addition, the chimera expresses only the N-terminus (MRRR) of CCR2 (FIG. 7C) with the third loop region of CCR1 or the third extracellular loop (RRRM) of CCR2 with the N-terminus of CCR1 and the first and second loop regions. Saturation binding of the 4.40 A68G S230P antibody to the receptor did not show any significant binding at concentrations below 10 μg / mL (FIG. 7D). In contrast, saturation binding of 4.40 A68G S230P antibodies to chimeric receptors expressing the N-terminus and third loop regions of CCR1 and the first and second loop regions (RMMR) of CCR2 resulted in significant doses at concentrations greater than 0.001 μg / mL. Dependent binding was shown (FIG. 7E). Staining of FLAG on the N terminus by M1 antibody was evaluated to confirm receptor expression on all receptor transfectants (Sigma F3040).

Table 7. Epitope Mapping

Peptide ELISA

In addition, epitope binding to the first and / or second loops of CCR2 was also assessed using peptide ELISA. After washing the Reacti-Bind NEUTRAVIDIN ™ Coated, High Binding Capacity ELISA plate (Pierce; Rockford, Ill.) Three times with PBS / 0.05% Tween20, biotinylated CCR2 Loop 2 (SEQ ID NO: 129) or Loop 3 (SEQ ID NO: 129) No. 130) coated with 100 μl / well of 6 μg / ml peptide (AnaSpec; San Jose, CA). The plate was incubated for 1 hour and then washed three times. The CCR2 antagonist antibody 4.40.3 A68G S230P or other primary antibody, then diluted in PBS / 0.1% BSA / 0.05% Tween20, was added to the plate in serial dilutions, incubated for 1 hour, and so were the other controls. Plates were washed three times and 100 μl / well HRP-Mouse anti-Human IgG4 secondary antibody (Zymed; San Francisco, Calif.) Was added to all wells at a 1: 5000 dilution and incubated for 1 hour. Plates were washed three times, TMB substrate was added to all wells and incubated for about 30 minutes. The color reaction was stopped by 2M H ₂ SO ₄ and absorbance readings measured on a plate reader at 450 nM (FIG. 8). Antibodies comprising antibodies 4.9 and 4.40 were found to bind to the second extracellular loop peptide of the receptor and not to the third loop.

Example 9

Join selectivity

Selectivity of the 4.40 A68G S230P antibody against human CCR2 was shown in FIG. 9 by the absence of chemotactic inhibition induced by the closely related chemokine receptor, binding to CCR5 and by the CCR5 / CCR1 ligand MIP-1a. It was confirmed by (FIG. 3). Anti-CCR5 antibody (Pharmingen, catalog 555992) bound to cells expressing CCR5 (FIG. 9B), but CCR2 antibody 4.40.3 A68G S230P did not show binding to these same cells (FIG. 9A). During this study, 300.19 cells engineered to express CCR5 were incubated for 30 minutes with either CCR2 selective antibody, 4.40 A68G S230P or CCR5 selective antibody. Cells were then washed in FACS buffer (0.02% sodium azide in PBS, 2% heat inactivated fetal bovine serum) and analyzed for cell surface expression using a flow cytometer using standard methods.

Example 10

Calcium Mobilization

To determine if antibody 4.40 acts as a functional antagonist on CCR2 and does not possess any significant agonist properties, the effect of 4.40.3 on calcium recruitment of CCR2 transfected 300-19 cells was tested as previously described. (Gladue RP et al., J Biol Chem 278: 40473-40480 (2003)). Spin down human CCR2 transfected 300-19 cells and place in PTI buffer (HBSS (Gibco, Grand Island, NY, USA) containing 10 mM Hepes (Gibco) and 4.0 mM CaCl ₂ (Sigma; St. Louis, MO). Resuspended at 2 × 10 ⁶ cells / ml by loading cells into 2 μl of indo-1 AM (Molecular Probes; Eugene, Oregon) per ml (final 2 μM) and for 25 minutes at 37 ° C. The cells were then washed twice with PTI buffer and suspended at 1 × 10 ⁷ / ml Different concentrations of antibody or appropriate controls were added to the cells and incubated for 30 minutes at room temperature 1 mm square. To cuvette (Sarstedt, Germany), 1.8 ml of preheated PTI buffer was added with 200 μl of cell suspension Excitation of cells and fluorescence using equipment obtained from Photon Technology Corporation International (PTI; Laurensville, NJ) The machine was stopped and 20 μl of 100 nM MCP-1 (Peprotech, Rocky Hill, NJ) were added, after which the following reagents were added in this order to release and chelate total calcium: 20 μl of 18% Triton X-100; 20 μl of 3M Tris (pH 8.5); 20 μl of 0.5M EGTA (pH 8.5) (both purchased from Sigma) Antibody 4.40 inhibited the ability of MCP-1 to induce calcium mobilization in a dose response manner (FIG. 10). Similar results were obtained with a 4.40 A68G S230P antibody.

Example 11

Chemotaxis Suppression for CCL2 and CCL7

4.40 Chemotaxis of THP-1 cells against MCP-1 (CCL2) is shown in FIG. 3 to determine if chemotaxis inhibition by A68G S230P antibody is specific for MCP-1 or if it also applies to other CCR2 ligands. As compared to that for CCL3 (MIP-1a). In addition to the migration of CCR2 transfected 300-19 cells for another CCR2 ligand, CCL7 (MCP-3) in the presence of antibody 4.40 A68G S230P was also assessed using similar chemotaxis assay as described in Example 6. 4.40 A68G S230P antibodies both inhibited chemotaxis for the known CCR2 ligands, CCL2 (FIG. 3) and CCL7 (FIG. 11), but FIG. 3 shows chemotaxis against the CCR1 / CCR5 ligand CCL3 (MIP-1a). It did not block.

Example 12

Monocytes Actin  polymerization

Human whole blood Actin  polymerization

Monoclonal antibody 4.40 A68G S230P was also tested for its ability to inhibit actin polymerization in human whole blood as shown in FIG. 12. Blood was collected in EDTA vacuum collection tubes (VWR; Boston, Miami, USA) and then incubated with dilutions of antibodies or KLH controls for 30 minutes at room temperature. 10 μl of the buffer control or 100 nM MCP-1 (Peprotech; Rocky Hill, NJ) was added to a 48 well Corning plate (VWR). 100 μl of blood was then added with gentle mixing, 0.8 ml stop / elution reagent (10% FACS eluent (Becton Dickinson, San Jose, CA), 20% 16% paraformaldehyde (Electron Microscopy Science; Incubation for 40 seconds after the reaction was stopped by Fort Washington, Philadelphia, USA and 70% H ₂ O). Plates were incubated for 10 minutes at room temperature, spin down, cells were transferred to 96 well round bottom polypropylene plates and washed twice with PBS. 100 μl of PBS was added per well. 50 μl of staining reagent (10% 5 mg / ml lysophosphotidylcholine (Sigma) in 10 × HBSS, 80% 16% paraformaldehyde and 10% 6.6 μM NBD Palasidine (Molecular Probes; Eugene ( Oregon, USA)). Cells were stained for 1 hour at room temperature in the dark. After incubation, cells were washed twice with PBS containing 2% fetal bovine serum (Hyclone, Logan, Utah, USA) and fluorescence was quantified on FACSCAN (Becton Dickinson) gated in monocytes. As shown in FIG. 12, antibody 4.40 A68G S230P inhibited actin polymerization with an IC ₅₀ of 0.168 μg / ml.

Cynomolgus  Monkey full blood Actin  polymerization

Blood was collected in EDTA vacuum collection tubes (VWR; Boston, Miami, USA) and then incubated with dilutions of antibody or isotype controls for 30 minutes at room temperature. During this time, 100 nM MCP-1 (Peprotech; Rocky Hill, NJ; final concentration 10 nM) or buffer control (HBSS without Ca ⁺² or Mg ⁺² (Gibco; Grand Island, NY, USA) and 0.2% BSA 10 μl (Sigma; St. Louis, MO) was added to 48 well Corning plates (VWR). 100 μl of blood was then added with gentle mixing and incubated for 40 seconds. 0.8 ml stop / elution reagent (10% FACS eluent (Becton Dickinson; San Jose, CA), 20% 16% paraformaldehyde (Electron Microscopy Science; Port Washington, Philadelphia, USA) and 70% H ₂ O Reaction was stopped. Plates were incubated for 10 minutes, spin down and washed twice with PBS. Cells were then stained with staining reagents (10% 5 mg / ml lysophosphotidylcholine (Sigma), 80% 16% paraformaldehyde and 10% 6.6 μM NBD Palasidine (Molecular Probes; Eugene (10% HBSS)). Stained in the dark for 1 hour at room temperature. After incubation, cells were washed twice with PBS containing 2% fetal bovine serum (Hyclone, Logan, Utah, USA) and fluorescence was quantified on FACSCAN (Becton Dickinson) gated in activated monocytes. As shown in FIG. 13, antibody 4.40 A68G S230P inhibited total blood actin polymerization in cynomolgus monkeys with an IC ₅₀ of 0.49 μg / ml.

Example 13

Collagen I mRNA  Quantitative Analysis

Human liver stellate cell line in flasks containing DME medium supplemented with 10% deactivated FBS, 100 U / ml penicillin / 100 μg / ml streptomycin and 2 mM L-Gln (Invitrogen) at 37 ° C. in a 5% CO 2 wet incubator. , LI-90 (JCRB Cellbank, Japan) was cultured. Cells were incubated at 20,000 cells / well for 3 days in 96 well tissue culture plates and treated with 1000 nM MCP-1 (PeproTech) and various concentrations of 4.40 A68G S230P for 48 hours at 37 ° C. Culture medium was prepared and cells were eluted by addition of 100 μl of elution buffer supplemented with QUANTIGENE ™ Expression Kit (Panomics). Branched DNA analysis was performed according to the manufacturer's instructions. Briefly, samples of total RNA were loaded onto 96 well microtiter plates containing hybridization buffer and 50 fmol / μL Col1A1 / GAPDH probe set. The captured mRNA was incubated at 46 ° C. for 60 minutes to hybridize to branched DNA molecules containing alkaline phosphatase molecules. After further incubation with the chemiluminescent substrate at 46 ° C. for 30 minutes, luminescence was quantified by a luminometer (ARVOsx, Perkin Elmer, Mass.). The ratio of luminescence of Col1A1 to GAPDH was calculated and the data analyzed by Prism 4.0 software to determine the IC ₅₀ value (GraphPad). As shown in Figure 14, 4.40 A68G S230P inhibited collagen IAI mRNA synthesis in MCP-1 induced LI90 cells with an IC ₅₀ value of 0.89 μg / mL (6.2 nM).

Example 14

pERK phosphorylation

Fresh whole human blood was isolated from healthy volunteers. FACS analysis showed high levels of phosphorylation of extracellular signal-regulated kinase (pERK) in CD14 + monocytes 6 min after addition of MCP-1. 4.40 A68G S230P inhibited MCP-1 induced pERK phosphorylation in whole blood in a dose dependent manner (FIG. 15). PERK phosphorylation in monocytes after in vitro stimulation with MCP-1 could be used as a mechanism biomarker to facilitate PK / PD and translational pharmacology. The IC ₅₀ and IC ₉₀ values obtained for the 4.40 A68G S230P antibody in this whole blood assay were 0.44 μg / mL (2.9 nM) and 0.89 μg / ml (6.1 nM), respectively.

Example 15

Rat  Acute hepatitis model

The efficiency of the 4.40 A68G S230P antibody was also tested using an acute mouse ConA model of hepatitis. For this experiment, transgenic mice with mouse CCR2 replaced with human CCR2 were used because the 4.40 A68G S230P antibody does not recognize rodent CCR2. Animals received a single intraperitoneal injection of mAb at 0.1, 0.3 or 1.0 mg / kg in saline. IgG ₄ isotype control antibody was administered to individual groups. After 30 minutes, the animals were injected iv tail vein (0.1 mL) with 15 mg / kg ConA in pyrogenic saline. The control group was given saline without ConA. After 24 hours, blood samples were obtained and analyzed for plasma liver enzymes. As shown in FIG. 16, ALT and AST were significantly elevated in antibody controls to values close to 25,000 U / L. In contrast, mice treated with 0.5 and 1.0 mg / kg of 4.40 A68G S230P showed a significant reduction in their plasma liver enzymes by approximately 50% and 80%, respectively.

Example 17

Therapeutic Effects in Animal Disease Models

Inflammation

In vivo mammalian models of inflammation were used to test the therapeutic effects of human anti-CCR2 antibodies or antigen-binding fragments thereof, including 4.40 and 4.9 antibodies. Leukocyte infiltration was monitored by intradermal injection of a suitable animal such as rabbit, mouse, rat, guinea pig or rhesus macaques with a chemokine such as MCP-1 and an antibody or fragment thereof reactive with mammalian CCR2 such as 4.40 antibody (eg For example, Van Damme, J. et al., J. Exp. Med. 176: 59-65 (1992); Zachariae, COC et al., J. Exp. Med. 171: 2177-2182 (1990) Jose, PJ et al., J. Exp. Med. 179: 881-887 (1994). Skin biopsy tissue was assessed histologically for infiltration of leukocytes (eg, eosinophils and granulocytes). Antagonist anti-CCR2 antibodies were expected to reduce leukocyte infiltration compared to control animals.

Multiple sclerosis

In vivo mammalian models of multiple sclerosis were used to test the therapeutic effects of antibodies or fragments thereof, including 4.40 and 4.9 antibodies. Experimental autoimmune encephalomyelitis (EAE) is a T cell mediator of the central nervous system (CNS) that acts as an animal model for multiple sclerosis (MS). Inflammatory disease (see Steinman L, Neuron 24: 511-514 (1999)). Immunized by encephalitis causing substances MOG _{35 -55} peptide (pi) 0 days and after 7 days, Mycobacterium tuberculosis bekul tube (tuberculosis), H37Ra (Difco) containing 70 ㎍ incomplete Freud's adjuvant (IFA; Difco, Detroit, Michigan, USA) with encephalitis-inducing substance of the total 600 _{35 -55} ㎍ emulsified MOG subcutaneous (sc) injection by the (two places, reinforcement side) C57 / J129 for the active EAE by And / or C57BL / 6 mice were sensitized. On days 0 and 2 pi, each mouse was also administered 500 ng of pertussis toxin (PTX; List Biological Laboratories, Capbell, CA) intravenously (iv) via the tail vein. Some animals also received elevated doses of anti-CCR2 antagonist antibodies, such as 4.40 antibodies. Animals were evaluated daily for clinical symptoms and on the following scale: grade 0, not abnormal; First grade, weak tail; Grade 2, limp tail and hind limbs fragile; Grade 3, hind limb paralysis; Grade 4, quadriplegia; And grade 5, reflexes or death.

In addition, light microscopic studies were performed on glutaraldehyde / osmium fixed tissue samples obtained from eye nerves, cerebral, cerebellum and spinal cord. Tissue samples were dehydrated and 1 μm test pieces were cut and embedded in epoxy resin stained with toluidine blue. Inflammation, demyelination, Wallerian degeneration (WD) and remyelination were rated on a scale of 0 to 5 as previously described (Cannella et al., Proc Natl Acad Sci USA 95: 10100-10105 (1998)). Mice treated with anti-CCR2 antagonist antibodies were expected to exhibit a reduction in symptoms of clinical abnormalities, inflammation, demyelination and WD as compared to control animals. In addition, this reduction was expected to occur in a dose dependent manner.

Neuropathic  ache

In vivo mammalian models of neuropathic pain were used to test the therapeutic effects of antibodies or fragments thereof, including 4.40 and 4.9 antibodies. C57BL mice were divided into two groups. The experimental group received daily anti-CCR2 antagonist antibody, such as 4.40 antibody, via tail vein injection, but the second group did not. The two groups were tested at time intervals after the start of treatment with the following analysis:

Motor function (Rota - rod). Initially, mice were trained for motor function for 3 minutes at a speed of 10 rpm. For the test, the speed was set at 10 rpm for 60 minutes and subsequently raised to 600 rpm. The time for the mice to decrease after starting the ascension was recorded.

Hot plate . Mice were tamed in a hot plate apparatus with the temperature set at 45 ° C. for 2 minutes. Subsequently, the mouse Placed on a hot plate and the temperature was sequentially changed to 52.5 ° C. and 55.5 ° C. (cutoff set to 30 seconds) respectively, followed by 58.5 ° C. (cutoff set to 20 seconds). The time the mouse licks or jumps his foot is recorded.

Formalin test . For 4 days prior to testing, mice were acclimated for 2 hours daily on a test platform. On the study date, mice were placed on the test platform for 1 hour and subsequently administered with 10 μl of 2% formalin on the plantar side of the left foot. The time for the mice to lick or lift the injected foot was recorded over a 2-minute period at 50 minute intervals for 50 minutes.

Thermal and mechanical stimulation . Thermal sensitivity was assessed by measuring foot withdrawal latency for radioactive thermal stimulation (Hargreaves et al., Pain 32: 77-88 (1988)). Mechanical sensitivity was determined by von Frey filament corrected using the up and down paradigms (Chaplan et al., J. Neurosci. Methods 53, 55-63 (1994)).

Nerve injury . Mice were anesthetized with a mixture of ketamine (50 mg / kg, im) and medetomymidine (1 mg / kg, im). An incision was made just below the tibia parallel to the sciatic nerve. The nerves were exposed and any adherent tissue was removed from the nerves. Ligation was firmly ligated with 6-0 silk sutures around ⅓ to ½ of the diameter of the sciatic nerve. The muscle was sealed with a suture and the wound was sealed with a round clip. Nerve damage 15 days before and after the machine The mouse's response to stimulation was tested.

Mice treated with anti-CCR2 antagonist antibodies were expected to show symptomatic reduction of neuropathic pain compared to control mice.

TABLE 8 CDR Sequence Analysis (SEQ ID NO :)

American Type Culture Collection (ATCC) PTA-6979 20050916 American Type Culture Collection (ATCC) PTA-6980 20050916 American Type Culture Collection (ATCC) PTA-6981 20050916 American Type Culture Collection (ATCC) PTA-7341 20060131 American Type Culture Collection (ATCC) PTA-7342 20060131

                         SEQUENCE LISTING <110> Pfizer Inc   <120> CCR2 antibodies <130> PC032985 <160> 203 <170> PatentIn version 3.5 <210> 1 <211> 1350 <212> DNA <213> Homo sapiens <400> 1 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agttatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt caaacatatg atggaagaaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa aacgttgtat 240 ctgcaaatga acagactgag agctgaggac acggctgtgt attattgtgc gagagatcag 300 gcgtactgga agtactttga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 420 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 600 aagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 720 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 780 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 840 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 960 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1260 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1320 aagagcctct ccctgtctct gggtaaatga 1350 <210> 2 <211> 366 <212> DNA <213> Homo sapiens <400> 2 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agttatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt caaacatatg atggaagaaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa aacgttgtat 240 ctgcaaatga acagactgag agctgaggac acggctgtgt attattgtgc gagagatcag 300 gcgtactgga agtactttga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 3 <211> 15 <212> DNA <213> Homo sapiens <400> 3 agttatggca tgcac 15 <210> 4 <211> 51 <212> DNA <213> Homo sapiens <400> 4 gttcaaacat atgatggaag aaataaatac tatgcagact ccgtgaaggg c 51 <210> 5 <211> 39 <212> DNA <213> Homo sapiens <400> 5 gatcaggcgt actggaagta ctttgatgct tttgatatc 39 <210> 6 <211> 90 <212> DNA <213> Homo sapiens <400> 6 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt 90 <210> 7 <211> 42 <212> DNA <213> Homo sapiens <400> 7 tgggtccgcc aggctccagg caaggggctg gagtgggtgg ca 42 <210> 8 <211> 96 <212> DNA <213> Homo sapiens <400> 8 cgattcacca tctccagaga caattccaag aaaacgttgt atctgcaaat gaacagactg 60 agagctgagg acacggctgt gtattattgt gcgaga 96 <210> 9 <211> 33 <212> DNA <213> Homo sapiens <400> 9 tggggccaag ggacaatggt caccgtctct tca 33 <210> 10 <211> 449 <212> PRT <213> Homo sapiens <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Gln Thr Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gln Ala Tyr Trp Lys Tyr Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp         195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr     210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly         435 440 445 Lys      <210> 11 <211> 122 <212> PRT <213> Homo sapiens <400> 11 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Gln Thr Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gln Ala Tyr Trp Lys Tyr Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 12 <211> 5 <212> PRT <213> Homo sapiens <400> 12 Ser Tyr Gly Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Homo sapiens <400> 13 Val Gln Thr Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 14 <211> 13 <212> PRT <213> Homo sapiens <400> 14 Asp Gln Ala Tyr Trp Lys Tyr Phe Asp Ala Phe Asp Ile 1 5 10 <210> 15 <211> 30 <212> PRT <213> Homo sapiens <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser             20 25 30 <210> 16 <211> 14 <212> PRT <213> Homo sapiens <400> 16 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 <210> 17 <211> 32 <212> PRT <213> Homo sapiens <400> 17 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg             20 25 30 <210> 18 <211> 11 <212> PRT <213> Homo sapiens <400> 18 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 19 <211> 645 <212> DNA <213> Homo sapiens <400> 19 gacatccaga tgacccagtc tccatcctcc ctgtctacat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacaatt ccccgtgcag ttttggccag 300 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa gaacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 20 <211> 321 <212> DNA <213> Homo sapiens <400> 20 gacatccaga tgacccagtc tccatcctcc ctgtctacat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacaatt ccccgtgcag ttttggccag 300 gggaccaagc tggagatcaa a 321 <210> 21 <211> 33 <212> DNA <213> Homo sapiens <400> 21 cgggcaagtc agagcattag cagctattta aat 33 <210> 22 <211> 21 <212> DNA <213> Homo sapiens <400> 22 gctgcatcca gtttgcaaag t 21 <210> 23 <211> 27 <212> DNA <213> Homo sapiens <400> 23 caacagagtt acaattcccc gtgcagt 27 <210> 24 <211> 69 <212> DNA <213> Homo sapiens <400> 24 gacatccaga tgacccagtc tccatcctcc ctgtctacat ctgtaggaga cagagtcacc 60 atcacttgc 69 <210> 25 <211> 45 <212> DNA <213> Homo sapiens <400> 25 tggtatcagc agaaaccagg gaaagcccct aagctcctga tctat 45 <210> 26 <211> 96 <212> DNA <213> Homo sapiens <400> 26 ggggtcccat caaggttcag tggcagtgga tctgggacag atttcactct caccatcagc 60 agtctgcaac ctgaagattt tgcaacttac tactgt 96 <210> 27 <211> 30 <212> DNA <213> Homo sapiens <400> 27 tttggccagg ggaccaagct ggagatcaaa 30 <210> 28 <211> 214 <212> PRT <213> Homo sapiens <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Ser Pro Cys                 85 90 95 Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Lys Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 29 <211> 107 <212> PRT <213> Homo sapiens <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Ser Pro Cys                 85 90 95 Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 30 <211> 11 <212> PRT <213> Homo sapiens <400> 30 Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 31 <211> 7 <212> PRT <213> Homo sapiens <400> 31 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Homo sapiens <400> 32 Gln Gln Ser Tyr Asn Ser Pro Cys Ser 1 5 <210> 33 <211> 23 <212> PRT <213> Homo sapiens <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys             20 <210> 34 <211> 15 <212> PRT <213> Homo sapiens <400> 34 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 35 <211> 32 <212> PRT <213> Homo sapiens <400> 35 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys             20 25 30 <210> 36 <211> 10 <212> PRT <213> Homo sapiens <400> 36 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 37 <211> 1350 <212> DNA <213> Homo sapiens <400> 37 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tgcactggat acgacaggcc 120 cctggacaag ggcttgagtg gatgggaatg atcaacccta gtggtggtag cacaacctac 180 gcacagaagt tccagggcag agtcaccttg accagggaca cgtccacgag cacagtctac 240 atggacctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagagaga 300 tggtataagt ggaacttcga tgtttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 420 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 600 aagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 720 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 780 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 840 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 960 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1260 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1320 aagagcctct ccctgtctct gggtaaatga 1350 <210> 38 <211> 366 <212> DNA <213> Homo sapiens <400> 38 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tgcactggat acgacaggcc 120 cctggacaag ggcttgagtg gatgggaatg atcaacccta gtggtggtag cacaacctac 180 gcacagaagt tccagggcag agtcaccttg accagggaca cgtccacgag cacagtctac 240 atggacctga gcagcctgag atctgaggac acggccgtat attactgtgc gagagagaga 300 tggtataagt ggaacttcga tgtttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 39 <211> 15 <212> DNA <213> Homo sapiens <400> 39 agctactata tgcac 15 <210> 40 <211> 51 <212> DNA <213> Homo sapiens <400> 40 atgatcaacc ctagtggtgg tagcacaacc tacgcacaga agttccaggg c 51 <210> 41 <211> 39 <212> DNA <213> Homo sapiens <400> 41 gagagatggt ataagtggaa cttcgatgtt tttgatatc 39 <210> 42 <211> 90 <212> DNA <213> Homo sapiens <400> 42 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc 90 <210> 43 <211> 42 <212> DNA <213> Homo sapiens <400> 43 tggatacgac aggcccctgg acaagggctt gagtggatgg ga 42 <210> 44 <211> 96 <212> DNA <213> Homo sapiens <400> 44 agagtcacct tgaccaggga cacgtccacg agcacagtct acatggacct gagcagcctg 60 agatctgagg acacggccgt atattactgt gcgaga 96 <210> 45 <211> 33 <212> DNA <213> Homo sapiens <400> 45 tggggccaag ggacaatggt caccgtctct tca 33 <210> 46 <211> 449 <212> PRT <213> Homo sapiens <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Met His Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Met Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Arg Trp Tyr Lys Trp Asn Phe Asp Val Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp         195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr     210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly         435 440 445 Lys      <210> 47 <211> 122 <212> PRT <213> Homo sapiens <400> 47 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Met His Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Met Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Arg Trp Tyr Lys Trp Asn Phe Asp Val Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 48 <211> 5 <212> PRT <213> Homo sapiens <400> 48 Ser Tyr Tyr Met His 1 5 <210> 49 <211> 17 <212> PRT <213> Homo sapiens <400> 49 Met Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly      <210> 50 <211> 13 <212> PRT <213> Homo sapiens <400> 50 Glu Arg Trp Tyr Lys Trp Asn Phe Asp Val Phe Asp Ile 1 5 10 <210> 51 <211> 30 <212> PRT <213> Homo sapiens <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr             20 25 30 <210> 52 <211> 14 <212> PRT <213> Homo sapiens <400> 52 Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 53 <211> 32 <212> PRT <213> Homo sapiens <400> 53 Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Asp 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg             20 25 30 <210> 54 <211> 11 <212> PRT <213> Homo sapiens <400> 54 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 55 <211> 648 <212> DNA <213> Homo sapiens <400> 55 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgca ggtctagtca aagccccgta tacagtgatg gaaacaccta cttgaattgg 120 cttcagcaga ggccaggcca atctccaagg cgcctaattt ataaggtttc taactgggac 180 gctggggtcc cagacagatt cagcggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttgggttt tattacactg gccgatcgat caccgtcggc 300 caagggacac gactggagat taaacgaact gtggctgcac catctgtctt catcttcccg 360 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttag 648 <210> 56 <211> 324 <212> DNA <213> Homo sapiens <400> 56 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgca ggtctagtca aagccccgta tacagtgatg gaaacaccta cttgaattgg 120 cttcagcaga ggccaggcca atctccaagg cgcctaattt ataaggtttc taactgggac 180 gctggggtcc cagacagatt cagcggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttgggttt tattacactg gccgatcgat caccgtcggc 300 caagggacac gactggagat taaa 324 <210> 57 <211> 48 <212> DNA <213> Homo sapiens <400> 57 aggtctagtc aaagccccgt atacagtgat ggaaacacct acttgaat 48 <210> 58 <211> 21 <212> DNA <213> Homo sapiens <400> 58 aaggtttcta actgggacgc t 21 <210> 59 <211> 18 <212> DNA <213> Homo sapiens <400> 59 actggccgat cgatcacc 18 <210> 60 <211> 69 <212> DNA <213> Homo sapiens <400> 60 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgc 69 <210> 61 <211> 45 <212> DNA <213> Homo sapiens <400> 61 tggcttcagc agaggccagg ccaatctcca aggcgcctaa tttat 45 <210> 62 <211> 93 <212> DNA <213> Homo sapiens <400> 62 ggggtcccag acagattcag cggcagtggg tcaggcactg atttcacact gaaaatcagc 60 agggtggagg ctgaggatgt tgggttttat tac 93 <210> 63 <211> 30 <212> DNA <213> Homo sapiens <400> 63 gtcggccaag ggacacgact ggagattaaa 30 <210> 64 <211> 215 <212> PRT <213> Homo sapiens <400> 64 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Pro Val Tyr Ser             20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser         35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Trp Asp Ala Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Thr Gly Arg Ser                 85 90 95 Ile Thr Val Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala             100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195 200 205 Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 65 <211> 108 <212> PRT <213> Homo sapiens <400> 65 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Pro Val Tyr Ser             20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser         35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Trp Asp Ala Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Thr Gly Arg Ser                 85 90 95 Ile Thr Val Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 <210> 66 <211> 16 <212> PRT <213> Homo sapiens <400> 66 Arg Ser Ser Gln Ser Pro Val Tyr Ser Asp Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 67 <211> 7 <212> PRT <213> Homo sapiens <400> 67 Lys Val Ser Asn Trp Asp Ala 1 5 <210> 68 <211> 6 <212> PRT <213> Homo sapiens <400> 68 Thr Gly Arg Ser Ile Thr 1 5 <210> 69 <211> 23 <212> PRT <213> Homo sapiens <400> 69 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys             20 <210> 70 <211> 15 <212> PRT <213> Homo sapiens <400> 70 Trp Leu Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr 1 5 10 15 <210> 71 <211> 31 <212> PRT <213> Homo sapiens <400> 71 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr             20 25 30 <210> 72 <211> 10 <212> PRT <213> Homo sapiens <400> 72 Val Gly Gln Gly Thr Arg Leu Glu Ile Lys 1 5 10 <210> 73 <211> 1350 <212> DNA <213> Homo sapiens <400> 73 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggact caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atattatatg atggaaagaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagatcag 300 gcgtactgga cctactttga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 420 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 600 aagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 720 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 780 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 840 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 960 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1260 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1320 aagagcctct ccctgtctct gggtaaatga 1350 <210> 74 <211> 366 <212> DNA <213> Homo sapiens <400> 74 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggact caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atattatatg atggaaagaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagatcag 300 gcgtactgga cctactttga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 75 <211> 15 <212> DNA <213> Homo sapiens <400> 75 agctatggca tgcac 15 <210> 76 <211> 51 <212> DNA <213> Homo sapiens <400> 76 gttatattat atgatggaaa gaataaatac tatgcagact ccgtgaaggg c 51 <210> 77 <211> 39 <212> DNA <213> Homo sapiens <400> 77 gatcaggcgt actggaccta ctttgatgct tttgatatc 39 <210> 78 <211> 90 <212> DNA <213> Homo sapiens <400> 78 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggact caccttcagt 90 <210> 79 <211> 42 <212> DNA <213> Homo sapiens <400> 79 tgggtccgcc aggctccagg caaggggctg gagtgggtgg ca 42 <210> 80 <211> 96 <212> DNA <213> Homo sapiens <400> 80 cgattcacca tctccagaga caattccaag aacacgctgt atctgcaaat gaacagcctg 60 agagctgagg acacggctgt gtattactgt gcgaga 96 <210> 81 <211> 33 <212> DNA <213> Homo sapiens <400> 81 tggggccaag ggacaatggt caccgtctct tca 33 <210> 82 <211> 449 <212> PRT <213> Homo sapiens <400> 82 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Leu Tyr Asp Gly Lys Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gln Ala Tyr Trp Thr Tyr Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp         195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr     210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly         435 440 445 Lys      <210> 83 <211> 122 <212> PRT <213> Homo sapiens <400> 83 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Leu Tyr Asp Gly Lys Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gln Ala Tyr Trp Thr Tyr Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 84 <211> 5 <212> PRT <213> Homo sapiens <400> 84 Ser Tyr Gly Met His 1 5 <210> 85 <211> 17 <212> PRT <213> Homo sapiens <400> 85 Val Ile Leu Tyr Asp Gly Lys Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 86 <211> 13 <212> PRT <213> Homo sapiens <400> 86 Asp Gln Ala Tyr Trp Thr Tyr Phe Asp Ala Phe Asp Ile 1 5 10 <210> 87 <211> 30 <212> PRT <213> Homo sapiens <400> 87 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser             20 25 30 <210> 88 <211> 14 <212> PRT <213> Homo sapiens <400> 88 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 <210> 89 <211> 32 <212> PRT <213> Homo sapiens <400> 89 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg             20 25 30 <210> 90 <211> 11 <212> PRT <213> Homo sapiens <400> 90 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 91 <211> 645 <212> DNA <213> Homo sapiens <400> 91 gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 atcacctgcc gggccagtca gagcattggt agtagcttac actggtacca gcagaaacca 120 gatcagtctc caaagctcct catcaagtat gcttcccagt ccttctcagg ggtcccctcg 180 aggttcagtg gcagtggatc tgcgacagat ttcaccctca ccatcaatag cctggaagct 240 gaagatgctg caacgtatta ctgtcatcag agtagtagtt taccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 92 <211> 321 <212> DNA <213> Homo sapiens <400> 92 gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 atcacctgcc gggccagtca gagcattggt agtagcttac actggtacca gcagaaacca 120 gatcagtctc caaagctcct catcaagtat gcttcccagt ccttctcagg ggtcccctcg 180 aggttcagtg gcagtggatc tgcgacagat ttcaccctca ccatcaatag cctggaagct 240 gaagatgctg caacgtatta ctgtcatcag agtagtagtt taccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 93 <211> 33 <212> DNA <213> Homo sapiens <400> 93 cgggccagtc agagcattgg tagtagctta cac 33 <210> 94 <211> 21 <212> DNA <213> Homo sapiens <400> 94 tatgcttccc agtccttctc a 21 <210> 95 <211> 27 <212> DNA <213> Homo sapiens <400> 95 catcagagta gtagtttacc gctcact 27 <210> 96 <211> 69 <212> DNA <213> Homo sapiens <400> 96 gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 atcacctgc 69 <210> 97 <211> 45 <212> DNA <213> Homo sapiens <400> 97 tggtaccagc agaaaccaga tcagtctcca aagctcctca tcaag 45 <210> 98 <211> 96 <212> DNA <213> Homo sapiens <400> 98 ggggtcccct cgaggttcag tggcagtgga tctgcgacag atttcaccct caccatcaat 60 agcctggaag ctgaagatgc tgcaacgtat tactgt 96 <210> 99 <211> 30 <212> DNA <213> Homo sapiens <400> 99 ttcggcggag ggaccaaggt ggagatcaaa 30 <210> 100 <211> 214 <212> PRT <213> Homo sapiens <400> 100 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser             20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 101 <211> 107 <212> PRT <213> Homo sapiens <400> 101 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser             20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 102 <211> 11 <212> PRT <213> Homo sapiens <400> 102 Arg Ala Ser Gln Ser Ile Gly Ser Ser Leu His 1 5 10 <210> 103 <211> 7 <212> PRT <213> Homo sapiens <400> 103 Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> 104 <211> 9 <212> PRT <213> Homo sapiens <400> 104 His Gln Ser Ser Ser Leu Pro Leu Thr 1 5 <210> 105 <211> 23 <212> PRT <213> Homo sapiens <400> 105 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys             20 <210> 106 <211> 15 <212> PRT <213> Homo sapiens <400> 106 Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys 1 5 10 15 <210> 107 <211> 32 <212> PRT <213> Homo sapiens <400> 107 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Ala Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys             20 25 30 <210> 108 <211> 10 <212> PRT <213> Homo sapiens <400> 108 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 109 <211> 645 <212> DNA <213> Homo sapiens <400> 109 gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 atcacctgcc gggccagtca gagcattggt agtagcttac actggtacca gcagaaacca 120 gatcagtctc caaagctcct catcaagtat gcttcccagt ccttctcagg ggtcccctcg 180 aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct 240 gaagatgctg caacgtatta ctgtcatcag agtagtagtt taccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645 <210> 110 <211> 321 <212> DNA <213> Homo sapiens <400> 110 gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 atcacctgcc gggccagtca gagcattggt agtagcttac actggtacca gcagaaacca 120 gatcagtctc caaagctcct catcaagtat gcttcccagt ccttctcagg ggtcccctcg 180 aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct 240 gaagatgctg caacgtatta ctgtcatcag agtagtagtt taccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 111 <211> 96 <212> DNA <213> Homo sapiens <400> 111 ggggtcccct cgaggttcag tggcagtgga tctgggacag atttcaccct caccatcaat 60 agcctggaag ctgaagatgc tgcaacgtat tactgt 96 <210> 112 <211> 214 <212> PRT <213> Homo sapiens <400> 112 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser             20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 113 <211> 107 <212> PRT <213> Homo sapiens <400> 113 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser             20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 114 <211> 32 <212> PRT <213> Homo sapiens <400> 114 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys             20 25 30 <210> 115 <211> 1350 <212> DNA <213> Homo sapiens <400> 115 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggact caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atattatatg atggaaagaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagatcag 300 gcgtactgga cctactttga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 420 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 600 aagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagtccaaat atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggacca 720 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 780 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 840 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 960 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1260 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1320 aagagcctct ccctgtctct gggtaaatga 1350 <210> 116 <211> 449 <212> PRT <213> Homo sapiens <400> 116 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Leu Tyr Asp Gly Lys Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gln Ala Tyr Trp Thr Tyr Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp         195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr     210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly         435 440 445 Lys      <210> 117 <211> 389 <212> PRT <213> Homo sapiens <400> 117 Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser Asp Tyr             20 25 30 Lys Asp Asp Asp Asp Val Asp Glu Thr Pro Asn Thr Thr Glu Asp Tyr         35 40 45 Asp Thr Thr Thr Glu Phe Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys     50 55 60 Val Asn Glu Arg Ala Phe Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser 65 70 75 80 Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val Leu Ile                 85 90 95 Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr Leu Leu             100 105 110 Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro Leu Trp         115 120 125 Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met Cys Lys     130 135 140 Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe 145 150 155 160 Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val                 165 170 175 Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr Ser Val             180 185 190 Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile Ile Phe         195 200 205 Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro Tyr Phe     210 215 220 Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile Leu Gly 225 230 235 240 Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly Ile Leu                 245 250 255 Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val             260 265 270 Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro         275 280 285 Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Asp Phe Leu Phe Thr     290 295 300 His Glu Cys Glu Gln Ser Arg His Leu Asp Leu Ala Val Gln Val Thr 305 310 315 320 Glu Val Ile Ala Tyr Thr His Cys Cys Val Asn Pro Val Ile Tyr Ala                 325 330 335 Phe Val Gly Glu Arg Phe Arg Lys Tyr Leu Arg Gln Leu Phe His Arg             340 345 350 Arg Val Ala Val His Leu Val Lys Trp Leu Pro Phe Leu Ser Val Asp         355 360 365 Arg Leu Glu Arg Val Ser Ser Thr Ser Pro Ser Thr Gly Glu His Glu     370 375 380 Leu Ser Ala Gly Phe 385 <210> 118 <211> 1167 <212> DNA <213> Homo sapiens <400> 118 atggacagca aaggttcgtc gcagaaaggg tcccgcctgc tcctgctgct ggtggtgtca 60 aatctactct tgtgccaggg tgtggtctcc gattacaaag atgatgatga tgtcgacgaa 120 actccaaaca ccacagagga ctatgacacg accacagagt ttgactatgg ggatgcaact 180 ccgtgccaga aggtgaacga gagggccttt ggggcccaac tcctgcctcc gctctactcg 240 ctggtgttca tctttggttt tgtgggcaac atgctggtcg tcctcatctt aataaactgc 300 aaaaagctga agtgcttgac tgacatttac ctgctcaacc tggccatctc tgatctgctt 360 tttcttatta ctctcccatt gtgggctcac tctgctgcaa atgagtgggt ctttgggaat 420 gcaatgtgca aattattcac agggctgtat cacatcggtt attttggcgg aatcttcttc 480 atcatcctcc tgacaatcga tagatacctg gctattgtcc atgctgtgtt tgctttaaaa 540 gccaggacgg tcacctttgg ggtggtgaca agtgtgatca cctggttggt ggctgtgttt 600 gcttctgtcc caggaatcat ctttactaaa tgccagaaag aagattctgt ttatgtctgt 660 ggcccttatt ttccacgagg atggaataat ttccacacaa taatgaggaa cattttgggg 720 ctggtcctgc cgctgctcat catggtcatc tgctactcgg gaatcctgaa aaccctgctt 780 cggtgtcgaa acgagaagaa gaggcatagg gcagtgagag tcatcttcac catcatgatt 840 gtttactttc tcttctggac tccctataac attgtcattc tcctgaacac cttccaagac 900 ttcctgttca cccatgagtg tgagcagagc agacatttgg acctggctgt gcaagtgacg 960 gaggtgatcg cctacacgca ctgctgtgtc aacccagtga tctacgcctt cgttggtgag 1020 aggttccgga agtacctgcg gcagttgttc cacaggcgtg tggctgtgca cctggttaaa 1080 tggctcccct tcctctccgt ggacaggctg gagagggtca gctccacatc tccctccaca 1140 ggggagcatg aactctctgc tgggttc 1167 <210> 119 <211> 394 <212> PRT <213> Homo sapiens <400> 119 Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser Asp Tyr             20 25 30 Lys Asp Asp Asp Asp Val Asp Glu Thr Pro Asn Thr Thr Glu Asp Tyr         35 40 45 Asp Thr Thr Thr Glu Phe Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys     50 55 60 Val Asn Glu Arg Ala Phe Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser 65 70 75 80 Leu Val Phe Val Ile Gly Leu Val Gly Asn Ile Leu Val Val Leu Val                 85 90 95 Leu Val Gln Tyr Lys Arg Leu Lys Asn Met Thr Ser Ile Tyr Leu Leu             100 105 110 Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Phe Thr Leu Pro Phe Trp         115 120 125 Ile Asp Tyr Lys Leu Lys Asp Asp Trp Val Phe Gly Asp Ala Met Cys     130 135 140 Lys Ile Leu Ser Gly Phe Tyr Tyr Thr Gly Leu Tyr Ser Glu Ile Phe 145 150 155 160 Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala                 165 170 175 Val Phe Ala Leu Arg Ala Arg Thr Val Thr Phe Gly Val Ile Thr Ser             180 185 190 Ile Ile Ile Trp Ala Leu Ala Ile Leu Ala Ser Met Pro Gly Leu Tyr         195 200 205 Phe Ser Lys Thr Gln Trp Glu Phe Thr His His Thr Cys Ser Leu His     210 215 220 Phe Pro His Glu Ser Leu Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys 225 230 235 240 Leu Asn Leu Phe Gly Leu Val Leu Pro Leu Leu Val Met Ile Ile Cys                 245 250 255 Tyr Thr Gly Ile Ile Lys Ile Leu Leu Arg Arg Pro Asn Glu Lys Lys             260 265 270 Ser Lys Ala Val Arg Leu Ile Phe Val Ile Met Ile Ile Phe Phe Leu         275 280 285 Phe Trp Thr Pro Tyr Asn Leu Thr Ile Leu Ile Ser Val Phe Gln Glu     290 295 300 Phe Phe Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala 305 310 315 320 Thr Gln Val Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro                 325 330 335 Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Val             340 345 350 Phe Phe Arg Lys His Ile Thr Lys Arg Phe Cys Lys Gln Cys Pro Val         355 360 365 Phe Tyr Arg Glu Thr Val Asp Gly Val Thr Ser Thr Asn Thr Pro Ser     370 375 380 Thr Gly Glu Gln Glu Val Ser Ala Gly Leu 385 390 <210> 120 <211> 1182 <212> DNA <213> Homo sapiens <400> 120 atggacagca aaggttcgtc gcagaaaggg tcccgcctgc tcctgctgct ggtggtgtca 60 aatctactct tgtgccaggg tgtggtctcc gattacaaag atgatgatga tgtcgacgaa 120 actccaaaca ccacagagga ctatgacacg accacagagt ttgactatgg ggatgcaact 180 ccgtgccaga aggtgaacga gagggccttt ggggcccaac tgctgccccc tctgtactcc 240 ttggtatttg tcattggcct ggttggaaac atcctggtgg tcctggtcct tgtgcaatac 300 aagaggctaa aaaacatgac cagcatctac ctcctgaacc tggccatttc tgacctgctc 360 ttcctgttca cgcttccctt ctggatcgac tacaagttga aggatgactg ggtttttggt 420 gatgccatgt gtaagatcct ctctgggttt tattacacag gcttgtacag cgagatcttt 480 ttcatcatcc tgctgacgat tgacaggtac ctggccatcg tccacgccgt gtttgccttg 540 cgggcacgga ccgtcacttt tggtgtcatc accagcatca tcatttgggc cctggccatc 600 ttggcttcca tgccaggctt atacttttcc aagacccaat gggaattcac tcaccacacc 660 tgcagccttc actttcctca cgaaagccta cgagagtgga agctgtttca ggctctgaaa 720 ctgaacctct ttgggctggt attgcctttg ttggtcatga tcatctgcta cacagggatt 780 ataaagattc tgctaagacg accaaatgag aagaaatcca aagctgtccg tttgattttt 840 gtcatcatga tcatcttttt tctcttttgg accccctaca atttgactat acttatttct 900 gttttccagg aattcttcgg cctgagtaac tgtgaaagca ccagtcaact ggaccaagcc 960 acgcaggtga cagagactct tgggatgact cactgctgca tcaatcccat catctatgcc 1020 ttcgttgggg agaagttcag aaggtatctc tcggtgttct tccgaaagca catcaccaag 1080 cgcttctgca aacaatgtcc agttttctac agggagacag tggatggagt gacttcaaca 1140 aacacgcctt ccactgggga gcaggaagtc tcggctggtt ta 1182 <210> 121 <211> 390 <212> PRT <213> Homo sapiens <400> 121 Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser Asp Tyr             20 25 30 Lys Asp Asp Asp Asp Val Asp Glu Thr Pro Asn Thr Thr Glu Asp Tyr         35 40 45 Asp Thr Thr Thr Glu Phe Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys     50 55 60 Val Asn Glu Arg Ala Phe Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser 65 70 75 80 Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val Leu Ile                 85 90 95 Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr Leu Leu             100 105 110 Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro Leu Trp         115 120 125 Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met Cys Lys     130 135 140 Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile Phe Phe 145 150 155 160 Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val                 165 170 175 Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr Ser Val             180 185 190 Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile Ile Phe         195 200 205 Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro Tyr Phe     210 215 220 Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile Leu Gly 225 230 235 240 Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly Ile Leu                 245 250 255 Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val             260 265 270 Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Thr Pro         275 280 285 Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Gly Leu     290 295 300 Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gln Val Thr 305 310 315 320 Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile Tyr Ala                 325 330 335 Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Val Phe Phe Arg Lys             340 345 350 His Ile Thr Lys Arg Phe Cys Lys Gln Cys Pro Val Phe Tyr Arg Glu         355 360 365 Thr Val Asp Gly Val Thr Ser Ser Thr Asn Thr Pro Ser Thr Gly Glu Gln     370 375 380 Glu Val Ser Ala Gly Leu 385 390 <210> 122 <211> 1170 <212> DNA <213> Homo sapiens <400> 122 atggacagca aaggttcgtc gcagaaaggg tcccgcctgc tcctgctgct ggtggtgtca 60 aatctactct tgtgccaggg tgtggtctcc gattacaaag atgatgatga tgtcgacgaa 120 actccaaaca ccacagagga ctatgacacg accacagagt ttgactatgg ggatgcaact 180 ccgtgccaga aggtgaacga gagggccttt ggggcccaac tcctgcctcc gctctactcg 240 ctggtgttca tctttggttt tgtgggcaac atgctggtcg tcctcatctt aataaactgc 300 aaaaagctga agtgcttgac tgacatttac ctgctcaacc tggccatctc tgatctgctt 360 tttcttatta ctctcccatt gtgggctcac tctgctgcaa atgagtgggt ctttgggaat 420 gcaatgtgca aattattcac agggctgtat cacatcggtt attttggcgg aatcttcttc 480 atcatcctcc tgacaatcga tagatacctg gctattgtcc atgctgtgtt tgctttaaaa 540 gccaggacgg tcacctttgg ggtggtgaca agtgtgatca cctggttggt ggctgtgttt 600 gcttctgtcc caggaatcat ctttactaaa tgccagaaag aagattctgt ttatgtctgt 660 ggcccttatt ttccacgagg atggaataat ttccacacaa taatgaggaa cattttgggg 720 ctggtcctgc cgctgctcat catggtcatc tgctactcgg gaatcctgaa aaccctgctt 780 cggtgtcgaa acgagaagaa gaggcatagg gcagtgagag tcatcttcac catcatgatt 840 gtttactttc tcttctggac tccctataac attgtcattc tcctgaacac cttccaggaa 900 ttcttcggcc tgagtaactg tgaaagcacc agtcaactgg accaagccac gcaggtgaca 960 gagactcttg ggatgactca ctgctgcatc aatcccatca tctatgcctt cgttggggag 1020 aagttcagaa ggtatctctc ggtgttcttc cgaaagcaca tcaccaagcg cttctgcaaa 1080 caatgtccag ttttctacag ggagacagtg gatggagtga cttcaacaaa cacgccttcc 1140 actggggagc aggaagtctc ggctggttta 1170 <210> 123 <211> 400 <212> PRT <213> Homo sapiens <400> 123 Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser Asp Tyr             20 25 30 Lys Asp Asp Asp Asp Val Asp Leu Ser Thr Ser Arg Ser Arg Phe Ile         35 40 45 Arg Asn Thr Asn Glu Ser Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr     50 55 60 Asp Tyr Gly Ala Pro Cys His Lys Phe Asp Val Lys Gln Ile Gly Ala 65 70 75 80 Gln Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val                 85 90 95 Gly Asn Met Leu Val Val Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys             100 105 110 Cys Leu Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu         115 120 125 Phe Leu Ile Thr Leu Pro Leu Trp Ala His Ser Ala Ala Asn Glu Trp     130 135 140 Val Phe Gly Asn Ala Met Cys Lys Leu Phe Thr Gly Leu Tyr His Ile 145 150 155 160 Gly Tyr Phe Gly Gly Ile Phe Ile Ile Leu Leu Thr Ile Asp Arg                 165 170 175 Tyr Leu Ala Ile Val His Ala Val Phe Ala Leu Arg Ala Arg Thr Val             180 185 190 Thr Phe Gly Val Ile Thr Ser Ile Ile Trp Ala Leu Ala Ile Leu         195 200 205 Ala Ser Met Pro Gly Leu Tyr Phe Ser Lys Thr Gln Trp Glu Phe Thr     210 215 220 His His Thr Cys Ser Leu His Phe Pro His Glu Ser Leu Arg Glu Trp 225 230 235 240 Lys Leu Phe Gln Ala Leu Lys Leu Asn Leu Phe Gly Leu Val Leu Pro                 245 250 255 Leu Leu Val Met Ile Ile Cys Tyr Thr Gly Ile Ile Lys Ile Leu Leu             260 265 270 Arg Arg Pro Asn Glu Lys Lys Ser Lys Ala Val Arg Leu Ile Phe Val         275 280 285 Ile Met Ile Ile Phe Phe Leu Phe Trp Thr Pro Tyr Asn Leu Thr Ile     290 295 300 Leu Ile Ser Val Phe Gln Asp Phe Leu Phe Thr His Glu Cys Glu Gln 305 310 315 320 Ser Arg His Leu Asp Leu Ala Val Gln Val Thr Glu Val Ile Ala Tyr                 325 330 335 Thr His Cys Cys Val Asn Pro Val Ile Tyr Ala Phe Val Gly Glu Arg             340 345 350 Phe Arg Lys Tyr Leu Arg Gln Leu Phe His Arg Arg Val Ala Val His         355 360 365 Leu Val Lys Trp Leu Pro Phe Leu Ser Val Asp Arg Leu Glu Arg Val     370 375 380 Ser Ser Thr Ser Pro Ser Thr Gly Glu His Glu Leu Ser Ala Gly Phe 385 390 395 400 <210> 124 <211> 1200 <212> DNA <213> Homo sapiens <400> 124 atggacagca aaggttcgtc gcagaaaggg tcccgcctgc tcctgctgct ggtggtgtca 60 aatctactct tgtgccaggg tgtggtctcc gattacaaag atgatgatga tgtcgacctg 120 tccacatctc gttctcggtt tatcagaaat accaacgaga gcggtgaaga agtcaccacc 180 ttttttgatt atgattacgg tgctccctgt cataaatttg acgtgaagca aattggggcc 240 caactcctgc ctccgctcta ctcgctggtg ttcatctttg gttttgtggg caacatgctg 300 gtcgtcctca tcttaataaa ctgcaaaaag ctgaagtgct tgactgacat ttacctgctc 360 aacctggcca tctctgatct gctttttctt attactctcc cattgtgggc tcactctgct 420 gcaaatgagt gggtctttgg gaatgcaatg tgcaaattat tcacagggct gtatcacatc 480 ggttattttg gcggaatctt tttcatcatc ctgctgacga ttgacaggta cctggccatc 540 gtccacgccg tgtttgcctt gcgggcacgg accgtcactt ttggtgtcat caccagcatc 600 atcatttggg ccctggccat cttggcttcc atgccaggct tatacttttc caagacccaa 660 tgggaattca ctcaccacac ctgcagcctt cactttcctc acgaaagcct acgagagtgg 720 aagctgtttc aggctctgaa actgaacctc tttgggctgg tattgccttt gttggtcatg 780 atcatctgct acacagggat tataaagatt ctgctaagac gaccaaatga gaagaaatcc 840 aaagctgtcc gtttgatttt tgtcatcatg atcatctttt ttctcttttg gaccccctac 900 aatttgacta tacttatttc tgttttccaa gacttcctgt tcacccatga gtgtgagcag 960 agcagacatt tggacctggc tgtgcaagtg acggaggtga tcgcctacac gcactgctgt 1020 gtcaacccag tgatctacgc cttcgttggt gagaggttcc ggaagtacct gcggcagttg 1080 ttccacaggc gtgtggctgt gcacctggtt aaatggctcc ccttcctctc cgtggacagg 1140 ctggagaggg tcagctccac atctccctcc acaggggagc atgaactctc tgctgggttc 1200 <210> 125 <211> 374 <212> PRT <213> Homo sapiens <400> 125 Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Ser 1 5 10 15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys             20 25 30 His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Leu         35 40 45 Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val     50 55 60 Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr 65 70 75 80 Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro                 85 90 95 Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met             100 105 110 Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile         115 120 125 Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His     130 135 140 Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr 145 150 155 160 Ser Val Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile                 165 170 175 Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro             180 185 190 Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile         195 200 205 Leu Gly Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly     210 215 220 Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg 225 230 235 240 Ala Val Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp                 245 250 255 Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe             260 265 270 Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gln         275 280 285 Val Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile     290 295 300 Tyr Ala Phe Val Gly Glu Lys Phe Arg Ser Leu Phe His Ile Ala Leu 305 310 315 320 Gly Cys Arg Ile Ala Pro Leu Gln Lys Pro Val Cys Gly Gly Pro Gly                 325 330 335 Val Arg Pro Gly Lys Asn Val Lys Val Thr Thr Gln Gly Leu Leu Asp             340 345 350 Gly Arg Gly Lys Gly Lys Ser Ile Gly Arg Ala Pro Glu Ala Ser Leu         355 360 365 Gln Asp Lys Glu Gly Ala     370 <210> 126 <211> 360 <212> PRT <213> Homo sapiens <400> 126 Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Ser 1 5 10 15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys             20 25 30 His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Leu         35 40 45 Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn Met Leu Val Val     50 55 60 Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Tyr 65 70 75 80 Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pro                 85 90 95 Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met             100 105 110 Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Ile         115 120 125 Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val His     130 135 140 Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe Gly Val Val Thr 145 150 155 160 Ser Val Ile Thr Trp Leu Val Ala Val Phe Ala Ser Val Pro Gly Ile                 165 170 175 Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro             180 185 190 Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg Asn Ile         195 200 205 Leu Gly Leu Val Leu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly     210 215 220 Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg 225 230 235 240 Ala Val Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp                 245 250 255 Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe             260 265 270 Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gln         275 280 285 Val Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile     290 295 300 Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Tyr Leu Ser Val Phe Phe 305 310 315 320 Arg Lys His Ile Thr Lys Arg Phe Cys Lys Gln Cys Pro Val Phe Tyr                 325 330 335 Arg Glu Thr Val Asp Gly Val Thr Ser Thr Asn Thr Pro Ser Thr Gly             340 345 350 Glu Gln Glu Val Ser Ala Gly Leu         355 360 <210> 127 <211> 42 <212> PRT <213> Homo sapiens <400> 127 Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Ser 1 5 10 15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cys             20 25 30 His Lys Phe Asp Val Lys Gln Ile Gly Ala         35 40 <210> 128 <211> 14 <212> PRT <213> Homo sapiens <400> 128 Ser Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Met Cys Lys 1 5 10 <210> 129 <211> 28 <212> PRT <213> Homo sapiens <400> 129 Thr Lys Cys Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pro Tyr Phe 1 5 10 15 Pro Arg Gly Trp Asn Asn Phe His Thr Ile Met Arg             20 25 <210> 130 <211> 17 <212> PRT <213> Homo sapiens <400> 130 Gln Glu Phe Phe Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp 1 5 10 15 Gln      <210> 131 <211> 352 <212> PRT <213> Homo sapiens <400> 131 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu             20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn         35 40 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met     50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe                 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe             100 105 110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu         115 120 125 Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe     130 135 140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr                 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn             180 185 190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu         195 200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys     210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu                 245 250 255 Leu Asn Thr Phe Gln Glu Phe Gly Leu Asn Asn Cys Ser Ser Ser             260 265 270 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu Gly Met Thr         275 280 285 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe     290 295 300 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser                 325 330 335 Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu             340 345 350 <210> 132 <211> 30 <212> DNA <213> Homo sapiens <400> 132 cgagagggcc tttgggatcc aactgctgcc 30 <210> 133 <211> 30 <212> DNA <213> Homo sapiens <133> 133 ggcagcagtt ggatcccaaa ggccctctcg 30 <210> 134 <211> 24 <212> DNA <213> Homo sapiens <400> 134 gcaaattggg atccaactcc tgcc 24 <210> 135 <400> 135 000 <210> 136 <211> 24 <212> DNA <213> Homo sapiens <400> 136 gcaaattggg gcccaactgc tgcc 24 <210> 137 <211> 24 <212> DNA <213> Homo sapiens <400> 137 ggcagcagtt gggccccaat ttgc 24 <210> 138 <211> 26 <212> DNA <213> Homo sapiens <400> 138 ctcttgtgcc agggtgtggt ctccga 26 <139> <211> 35 <212> DNA <213> Homo sapiens <400> 139 gatcgaagct ttcagaaccc agcagagagt tcatg 35 <210> 140 <211> 43 <212> DNA <213> Homo sapiens <400> 140 gactatactt atttctgttt tcattgtcat tctcctgaac acc 43 <210> 141 <211> 29 <212> DNA <213> Homo sapiens <400> 141 cgccaagctt cattataaac cagccgaga 29 <210> 142 <211> 31 <212> DNA <213> Homo sapiens <400> 142 acgcgtcgac gaaactccaa acaccacaga g 31 <210> 143 <211> 40 <212> DNA <213> Homo sapiens <400> 143 gttcaggaga atgacaatga aaacagaaat aagtatagtc 40 <210> 144 <211> 39 <212> DNA <213> Homo sapiens <400> 144 cattctcctg aacaccttcc aagacttcct gttcaccca 39 <210> 145 <211> 29 <212> DNA <213> Homo sapiens <400> 145 gccaagcttc cagtgtgatg gatatctga 29 <210> 146 <211> 40 <212> DNA <213> Homo sapiens <400> 146 tgggtgaaca ggaagtcttg gaaggtgttc aggagaatga 40 <210> 147 <211> 23 <212> DNA <213> Homo sapiens <400> 147 caggtgcagc tggtggagtc tgg 23 <210> 148 <211> 25 <212> DNA <213> Homo sapiens <400> 148 gaagagacgg tgaccattgt ccctt 25 <210> 149 <211> 27 <212> DNA <213> Homo sapiens <400> 149 gaaattgtgc tgactcagtc tccagac 27 <210> 150 <211> 24 <212> DNA <213> Homo sapiens <400> 150 gtttgatctc caccttggtc cctc 24 <210> 151 <211> 27 <212> DNA <213> Homo sapiens <400> 151 ggcagtggat ctgggacaga tttcacc 27 <210> 152 <211> 27 <212> DNA <213> Homo sapiens <400> 152 ggtgaaatct gtcccagatc cactgcc 27 <210> 153 <211> 88 <212> DNA <213> Homo sapiens <400> 153 ttatgctggg cccagctctg tcccacaccg cggtcacatg gcaccacctc tcttgcagct 60 tccaccaaag gcccatccgt cttccccc 88 <210> 154 <211> 39 <212> DNA <213> Homo sapiens <400> 154 tcatattctc tagatcattt acccagagac agggagagg 39 <210> 155 <211> 27 <212> DNA <213> Homo sapiens <400> 155 cagcgtggtg acagtgccct ccagcag 27 <210> 156 <211> 27 <212> DNA <213> Homo sapiens <400> 156 ctgctggagg gcactgtcac cacgctg 27 <210> 157 <211> 12754 <212> DNA <213> Homo sapiens <400> 157 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat 60 acataactga gaatagagaa gttcagatca aggttaggaa ggggtggaca tccaaaccgt 120 tcgatcgaat tcattgcggt aaaacgttcc gtacctttta tgtattgact cttatctctt 180 caagtctagt tccaatcctt cagagagaca gcagaatatg ggccaaacag gatatctgtg 240 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc 300 gtctctctgt cgtcttatac ccggtttgtc ctatagacac cattcgtcaa ggacggggcc 360 gagtcccggt tcttgtctac caggggtcta cgccagggcg cctcagcagt ttctagagaa 420 ccatcagatg tttccagggt gccccaagga cctgaaaatg accctgtgcc ttatttgaac 480 taaccaatca gttcgcttct ggagtcgtca aagatctctt ggtagtctac aaaggtccca 540 cggggttcct ggacttttac tgggacacgg aataaacttg attggttagt caagcgaaga 600 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact 660 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac gcgaagacaa gcgcgcgaag 720 acgaggggct cgagttattt tctcgggtgt tggggagtga gccgcgcggt caggaggcta 780 tctgacgcag cgggcccatg cccccccccc cccgtattcc caataaagcc tcttgctgtt 840 tgcatccgaa tcgtggactc gctgatcctt gggagggtct cctcagattg attgactgcc 900 gggggggggg gggcataagg gttatttcgg agaacgacaa acgtaggctt agcacctgag 960 cgactaggaa ccctcccaga ggagtctaac taactgacgg cacctcgggg gtctttcatt 1020 tggaggttcc accgagattt ggagacccct gcctagggac caccgacccc cccgccggga 1080 ggtaagctgg ccagcggtcg gtggagcccc cagaaagtaa acctccaagg tggctctaaa 1140 cctctgggga cggatccctg gtggctgggg gggcggccct ccattcgacc ggtcgccagc 1200 tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg 1260 tctgtactag ttagctaact agctctgtat ctggcggacc aaagcacaga cagagacaga 1320 aacacgcaca aacacggccg tagattacaa acgcggacgc agacatgatc aatcgattga 1380 tcgagacata gaccgcctgg cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc 1440 ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga ggaagggagt 1500 gcaccacctt gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg 1560 aaacccccgg caaaaacacc gggctggact ccttccctca cgatgtggaa tccgaccccg 1620 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa 1680 tttttgcttt cggtttggaa gctacacctt aggctggggc agtcctatac accaagacca 1740 tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa gccaaacctt 1800 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct 1860 gactgtgttt ctgtatttgt ctgaaaatta gggccagact ggcttcggcg cgcagaacag 1920 acgacgtcgc gacgtcgtag caagacacaa cagagacaga ctgacacaaa gacataaaca 1980 gacttttaat cccggtctga gttaccactc ccttaagttt gaccttaggt cactggaaag 2040 atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct 2100 caatggtgag ggaattcaaa ctggaatcca gtgacctttc tacagctcgc ctagcgagtg 2160 ttggtcagcc atctacagtt cttctctgca acccaatgga tctgctctgc agaatggcca 2220 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt 2280 aagatcaagg tcttttcacc agacgagacg tcttaccggt tggaaattgc agcctaccgg 2340 cgctctgccg tggaaattgg ctctggagta gtgggtccaa ttctagttcc agaaaagtgg 2400 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt 2460 tgacccccct ccctgggtca agccctttgt acaccctaag accgggcgta cctgtgggtc 2520 tggtccaggg gatgtagcac tggacccttc ggaaccgaaa actgggggga gggacccagt 2580 tcgggaaaca tgtgggattc cctccgcctc ctcttcctcc atccgccccg tctctccccc 2640 ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc 2700 ggaggcggag gagaaggagg taggcggggc agagaggggg aacttggagg agcaagctgg 2760 ggcggagcta ggagggaaat aggtcgggag tgaggaagag taggcgccga gatctctcga 2820 ggttaaactt aagctatgga cagcaaaggt tcgtcgcaga aagggtcccg cctgctcctg 2880 ctgctggtgg tgtcaaatct atccgcggct ctagagagct ccaatttgaa ttcgatacct 2940 gtcgtttcca agcagcgtct ttcccagggc ggacgaggac gacgaccacc acagtttaga 3000 actcttgtgc cagggtgtgg tctccgatta caaagatgat gatgatagcg ctgaagacaa 3060 taatatgtta cctcagttca tccacggcat actatcaaca tgagaacacg gtcccacacc 3120 agaggctaat gtttctacta ctactatcgc gacttctgtt attatacaat ggagtcaagt 3180 aggtgccgta tgatagttgt tctcattctc tatttacacg aagtatccaa gagcttgatg 3240 aaggggccac cacaccgtat gactacgatg atggtgagcc ttgtcataaa accagtgtga 3300 agagtaagag ataaatgtgc ttcataggtt ctcgaactac ttccccggtg gtgtggcata 3360 ctgatgctac taccactcgg aacagtattt tggtcacact agcaaattgg agcttggatc 3420 ctgcctccgc tctactcgct ggtgttcatc tttggttttg tgggcaacat gctggtcgtc 3480 ctcatcttaa taaactgcaa tcgtttaacc tcgaacctag gacggaggcg agatgagcga 3540 ccacaagtag aaaccaaaac acccgttgta cgaccagcag gagtagaatt atttgacgtt 3600 aaagctgaag tgcttgactg acatttacct gctcaacctg gccatctctg atctgctttt 3660 tcttattact ctcccattgt gggctcactc tgctgcaaat tttcgacttc acgaactgac 3720 tgtaaatgga cgagttggac cggtagagac tagacgaaaa agaataatga gagggtaaca 3780 cccgagtgag acgacgttta gagtgggtct ttgggaatgc aatgtgcaaa ttattcacag 3840 ggctgtatca catcggttat tttggcggaa tcttcttcat catcctcctg acaatcgata 3900 ctcacccaga aacccttacg ttacacgttt aataagtgtc ccgacatagt gtagccaata 3960 aaaccgcctt agaagaagta gtaggaggac tgttagctat gatacctggc tattgtccat 4020 gctgtgtttg ctttaaaagc caggacggtc acctttgggg tggtgacaag tgtgatcacc 4080 tggttggtgg ctgtgtttgc ctatggaccg ataacaggta cgacacaaac gaaattttcg 4140 gtcctgccag tggaaacccc accactgttc acactagtgg accaaccacc gacacaaacg 4200 ttctgtccca ggaatcatct ttactaaatg ccagaaagaa gattctgttt atgtctgtgg 4260 cccttatttt ccacgaggat ggaataattt ccacacaata aagacagggt ccttagtaga 4320 aatgatttac ggtctttctt ctaagacaaa tacagacacc gggaataaaa ggtgctccta 4380 ccttattaaa ggtgtgttat atgaggaaca ttttggggct ggtcctgccg ctgctcatca 4440 tggtcatctg ctactcggga atcctgaaaa ccctgcttcg gtgtcgaaac gagaagaaga 4500 tactccttgt aaaaccccga ccaggacggc gacgagtagt accagtagac gatgagccct 4560 taggactttt gggacgaagc cacagctttg ctcttcttct ggcatagggc agtgagagtc 4620 atcttcacca tcatgattgt ttactttctc ttctggactc cctataatat tgtcattctc 4680 ctgaacacct tccaggaatt ccgtatcccg tcactctcag tagaagtggt agtactaaca 4740 aatgaaagag aagacctgag ggatattata acagtaagag gacttgtgga aggtccttaa 4800 cttcggcctg agtaactgtg aaagcaccag tcaactggac caagccacgc aggtgacaga 4860 gactcttggg atgactcact gctgcatcaa tcccatcatc gaagccggac tcattgacac 4920 tttcgtggtc agttgacctg gttcggtgcg tccactgtct ctgagaaccc tactgagtga 4980 cgacgtagtt agggtagtag tatgccttcg ttggggagaa gttcagaagg tatctctcgg 5040 tgttcttccg aaagcacatc accaagcgct tctgcaaaca atgtccagtt ttctacaggg 5100 atacggaagc aacccctctt caagtcttcc atagagagcc acaagaaggc tttcgtgtag 5160 tggttcgcga agacgtttgt tacaggtcaa aagatgtccc agacagtgga tggagtgact 5220 tcaacaaaca cgccttccac tggggagcag gaagtctcgg ctggtttata atgactcgag 5280 ctctctagag ggcccgtttg tctgtcacct acctcactga agttgtttgt gcggaaggtg 5340 acccctcgtc cttcagagcc gaccaaatat tactgagctc gagagatctc ccgggcaaac 5400 acctgcagcc aagcttatcg ataaaataaa agattttatt tagtctccag aaaaaggggg 5460 gaatgaaaga ccccacctgt aggtttggca agctagctta tggacgtcgg ttcgaatagc 5520 tattttattt tctaaaataa atcagaggtc tttttccccc cttactttct ggggtggaca 5580 tccaaaccgt tcgatcgaat agtaacgcca ttttgcaagg catggaaaat acataactga 5640 gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag 5700 tcattgcggt aaaacgttcc gtacctttta tgtattgact cttatctctt caagtctagt 5760 tccaatcctt gtctctctgt cgtcttatac ccggtttgtc gatatctgtg gtaagcagtt 5820 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc cctcagcagt 5880 ttctagagaa ccatcagatg ctatagacac cattcgtcaa ggacggggcc gagtcccggt 5940 tcttgtctac caggggtcta cgccagggcg ggagtcgtca aagatctctt ggtagtctac 6000 tttccagggt gccccaagga cctgaaaatg accctgtgcc ttatttgaac taaccaatca 6060 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga aaaggtccca cggggttcct 6120 ggacttttac tgggacacgg aataaacttg attggttagt caagcgaaga gcgaagacaa 6180 gcgcgcgaag acgaggggct gctcaataaa agagcccaca acccctcact cggcgcgcca 6240 gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg 6300 cgagttattt tctcgggtgt tggggagtga gccgcgcggt caggaggcta tctgacgcag 6360 cgggcccatg ggcacatagg ttatttggga gaacgtcaac catccgactt gtggtctcgc 6420 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatgg 6480 gtaacagttt cttgaagttg gtaggctgaa caccagagcg acaaggaacc ctcccagagg 6540 agactcacta actgatgggc agtcgccccc agaaagtacc cattgtcaaa gaacttcaac 6600 gagaacaaca ttctgagggt aggagtcgaa tattaagtaa tcctgactca attagccact 6660 gttttgaatc cacatactcc aatactcctg aaatagttca ctcttgttgt aagactccca 6720 tcctcagctt ataattcatt aggactgagt taatcggtga caaaacttag gtgtatgagg 6780 ttatgaggac tttatcaagt ttatggacag cgcagaaaga gctggggaga attgtgaaat 6840 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 6900 aatacctgtc gcgtctttct cgacccctct taacacttta acaataggcg agtgttaagg 6960 tgtgttgtat gctcggcctt cgtatttcac atttcggacc ggtgcctaat gagtgagcta 7020 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 7080 gctgcattaa tgaatcggcc ccacggatta ctcactcgat tgagtgtaat taacgcaacg 7140 cgagtgacgg gcgaaaggtc agccctttgg acagcacggt cgacgtaatt acttagccgg 7200 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 7260 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc ttgcgcgccc ctctccgcca 7320 aacgcataac ccgcgagaag gcgaaggagc gagtgactga gcgacgcgag ccagcaagcc 7380 gacgccgctc gccatagtcg tcactcaaag gcggtaatac ggttatccac agaatcaggg 7440 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 7500 agtgagtttc cgccattatg ccaataggtg tcttagtccc ctattgcgtc ctttcttgta 7560 cactcgtttt ccggtcgttt tccggtcctt ggcatttttc gccgcgttgc tggcgttttt 7620 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7680 aaacccgaca ggactataaa cggcgcaacg accgcaaaaa ggtatccgag gcggggggac 7740 tgctcgtagt gtttttagct gcgagttcag tctccaccgc tttgggctgt cctgatattt 7800 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 7860 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ctatggtccg caaaggggga 7920 ccttcgaggg agcacgcgag aggacaaggc tgggacggcg aatggcctat ggacaggcgg 7980 aaagagggaa gcccttcgca ggcgctttct catagctcac gctgtaggta tctcagttcg 8040 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 8100 ccgcgaaaga gtatcgagtg cgacatccat agagtcaagc cacatccagc aagcgaggtt 8160 cgacccgaca cacgtgcttg gggggcaagt cgggctggcg tgcgccttat ccggtaacta 8220 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 8280 caggattagc agagcgaggt acgcggaata ggccattgat agcagaactc aggttgggcc 8340 attctgtgct gaatagcggt gaccgtcgtc ggtgaccatt gtcctaatcg tctcgctcca 8400 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 8460 cagtatttgg tatctgcgct ctgctgaagc cagttacctt tacatccgcc acgatgtctc 8520 aagaacttca ccaccggatt gatgccgatg tgatcttcct gtcataaacc atagacgcga 8580 gacgacttcg gtcaatggaa cggaaaaaga gttggtagct cttgatccgg caaacaaacc 8640 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 8700 gcctttttct caaccatcga gaactaggcc gtttgtttgg tggcgaccat cgccaccaaa 8760 aaaacaaacg ttcgtcgtct aatgcgcgtc tttttttcct tctcaagaag atcctttgat 8820 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 8880 gagattatca aaaaggatct agagttcttc taggaaacta gaaaagatgc cccagactgc 8940 gagtcacctt gcttttgagt gcaattccct aaaaccagta ctctaatagt ttttcctaga 9000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 9060 aaacttggtc tgacagttac caatgcttaa tcagtgaggc agtggatcta ggaaaattta 9120 atttttactt caaaatttag ttagatttca tatatactca tttgaaccag actgtcaatg 9180 gttacgaatt agtcactccg acctatctca gcgatctgtc tatttcgttc atccatagtt 9240 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 9300 tggatagagt cgctagacag ataaagcaag taggtatcaa cggactgagg ggcagcacat 9360 ctattgatgc tatgccctcc cgaatggtag accggggtca gctgcaatga taccgcgaga 9420 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 9480 cagaagtggt cctgcaactt cgacgttact atggcgctct gggtgcgagt ggccgaggtc 9540 taaatagtcg ttatttggtc ggtcggcctt cccggctcgc gtcttcacca ggacgttgaa 9600 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 9660 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat ataggcggag gtaggtcaga 9720 taattaacaa cggcccttcg atctcattca tcaagcggtc aattatcaaa cgcgttgcaa 9780 caacggtaac gatgtccgta cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 9840 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 9900 gcaccacagt gcgagcagca aaccataccg aagtaagtcg aggccaaggg ttgctagttc 9960 cgctcaatgt actagggggt acaacacgtt ttttcgccaa agctccttcg gtcctccgat 10020 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 10080 ttctcttact gtcatgccat tcgaggaagc caggaggcta gcaacagtct tcattcaacc 10140 ggcgtcacaa tagtgagtac caataccgtc gtgacgtatt aagagaatga cagtacggta 10200 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 10260 tgcggcgacc gagttgctct tgcccggcgt caatacggga ggcattctac gaaaagacac 10320 tgaccactca tgagttggtt cagtaagact cttatcacat acgccgctgg ctcaacgaga 10380 acgggccgca gttatgccct taataccgcg ccacatagca gaactttaaa agtgctcatc 10440 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 10500 attatggcgc ggtgtatcgt cttgaaattt tcacgagtag taaccttttg caagaagccc 10560 cgcttttgag agttcctaga atggcgacaa ctctaggtca tcgatgtaac ccactcgtgc 10620 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 10680 aaggcaaaat gccgcaaaaa agctacattg ggtgagcacg tgggttgact agaagtcgta 10740 gaaaatgaaa gtggtcgcaa agacccactc gtttttgtcc ttccgtttta cggcgttttt 10800 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 10860 gaagcattta tcagggttat tgtctcatga gcggatacat tcccttattc ccgctgtgcc 10920 tttacaactt atgagtatga gaaggaaaaa gttataataa cttcgtaaat agtcccaata 10980 acagagtact cgcctatgta atttgaatgt atttagaaaa ataaacaaat aggggttccg 11040 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 11100 taaacttaca taaatctttt tatttgttta tccccaaggc gcgtgtaaag gggcttttca 11160 cggtggactg cagattcttt ggtaataata gtactgtaat acctataaaa ataggcgtat 11220 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 11280 gctcccggag acggtcacag tggatatttt tatccgcata gtgctccggg aaagcagagc 11340 gcgcaaagcc actactgcca cttttggaga ctgtgtacgt cgagggcctc tgccagtgtc 11400 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 11460 gcgggtgtcg gggctggctt aactatgcgg catcagagca gaacagacat tcgcctacgg 11520 ccctcgtctg ttcgggcagt cccgcgcagt cgcccacaac cgcccacagg cccgaccgaa 11580 ttgatacgcc gtagtctcgt gattgtactg agagtgcacc atatgcggtg tgaaataccg 11640 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac 11700 ctaacatgac tctcacgtgg tatacgccac actttatggc gtgtctacgc attcctcttt 11760 tatggcgtag tccgcggtaa gcggtaagtc cgacgcgttg tgttgggaag ggcgatcggt 11820 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 11880 ttgggtaacg ccagggtttt acaacccttc ccgctagcca cgcccggaga agcgataatg 11940 cggtcgaccg ctttccccct acacgacgtt ccgctaattc aacccattgc ggtcccaaaa 12000 cccagtcacg acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg agatggcgcc 12060 caacagtccc ccggccacgg ggcctgccac catacccacg gggtcagtgc tgcaacattt 12120 tgctgccgcg ttccttacca cgtacgttcc tctaccgcgg gttgtcaggg ggccggtgcc 12180 ccggacggtg gtatgggtgc ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat 12240 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga 12300 ggctttgttc gcgagtactc gggcttcacc gctcgggcta gaaggggtag ccactacagc 12360 cgctatatcc gcggtcgttg gcgtggacac cgcggccact tgccggccac gatgcgtccg 12420 gcgtagaggc gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt 12480 ttgactcaac aatatcacca acggccggtg ctacgcaggc cgcatctccg ctaatcaggt 12540 taaacaattt ctgtcctata gtcaccaggt ccgagatcaa aactgagttg ttatagtggt 12600 gctgaagcct atagagtacg agccatagat aaaataaaag attttattta gtctccagaa 12660 aaagggggga tgaaagacga cttcggatat ctcatgctcg gtatctattt tattttctaa 12720 aataaatcag aggtcttttt cccccctact ttct 12754 <210> 158 <211> 119 <212> PRT <213> Homo sapiens <400> 158 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Tyr Asn Trp Asn Tyr Ala Phe Asp Ile Trp Gly Gln Gly             100 105 110 Thr Met Val Thr Val Ser Ser         115 <210> 159 <211> 112 <212> PRT <213> Homo sapiens <400> 159 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser             20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser         35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Trp Asp Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly                 85 90 95 Thr His Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys             100 105 110 <210> 160 <211> 118 <212> PRT <213> Homo sapiens <400> 160 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Tyr Asn Trp Asn Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr             100 105 110 Met Val Thr Val Ser Ser         115 <210> 161 <211> 107 <212> PRT <213> Homo sapiens <400> 161 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser             20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile         35 40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 162 <211> 117 <212> PRT <213> Homo sapiens <400> 162 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Gly             20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala         35 40 45 Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Tyr Asn Trp Asn Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met             100 105 110 Val Thr Val Ser Ser         115 <210> 163 <211> 107 <212> PRT <213> Homo sapiens <400> 163 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr             20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 164 <211> 118 <212> PRT <213> Homo sapiens <400> 164 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Tyr Asn Trp Asn Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr             100 105 110 Met Val Thr Val Ser Ser         115 <210> 165 <211> 113 <212> PRT <213> Homo sapiens <400> 165 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser             20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                 85 90 95 Tyr Tyr Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys      <210> 166 <211> 1350 <212> DNA <213> Homo sapiens <400> 166 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tacattgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaatg atcaatccta gtggtggtcg cacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggacctga gcagcctgag atctgaggac acggccgtgt tttactgtgc gagagagaga 300 tggtataagt ggaacttcga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcctcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 420 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 480 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540 tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 600 aagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt 660 gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 720 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 780 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 840 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 960 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1260 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1320 aagagcctct ccctgtctct gggtaaatga 1350 <210> 167 <211> 366 <212> DNA <213> Homo sapiens <400> 167 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tacattgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaatg atcaatccta gtggtggtcg cacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggacctga gcagcctgag atctgaggac acggccgtgt tttactgtgc gagagagaga 300 tggtataagt ggaacttcga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcctca 366 <210> 168 <211> 15 <212> DNA <213> Homo sapiens <400> 168 agctactata tacat 15 <210> 169 <211> 51 <212> DNA <213> Homo sapiens <400> 169 atgatcaatc ctagtggtgg tcgcacaagc tacgcacaga agttccaggg c 51 <210> 170 <211> 39 <212> DNA <213> Homo sapiens <400> 170 gagagatggt ataagtggaa cttcgatgct tttgatatc 39 <210> 171 <211> 90 <212> DNA <213> Homo sapiens <400> 171 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc 90 <210> 172 <211> 42 <212> DNA <213> Homo sapiens <400> 172 tgggtgcgac aggcccctgg acaagggctt gagtggatgg ga 42 <210> 173 <211> 96 <212> DNA <213> Homo sapiens <400> 173 agagtcacca tgaccaggga cacgtccacg agcacagtct acatggacct gagcagcctg 60 agatctgagg acacggccgt gttttactgt gcgaga 96 <210> 174 <211> 33 <212> DNA <213> Homo sapiens <400> 174 tggggccaag ggacaatggt caccgtctcc tca 33 <175> 175 <211> 449 <212> PRT <213> Homo sapiens <400> 175 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Met Ile Asn Pro Ser Gly Gly Pro Thr Ser Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Phe Tyr Cys                 85 90 95 Ala Arg Glu Arg Trp Tyr Lys Trp Asn Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp         195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr     210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly         435 440 445 Lys      <210> 176 <211> 122 <212> PRT <213> Homo sapiens <400> 176 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Met Ile Asn Pro Ser Gly Gly Pro Thr Ser Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Phe Tyr Cys                 85 90 95 Ala Arg Glu Arg Trp Tyr Lys Trp Asn Phe Asp Ala Phe Asp Ile Trp             100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser         115 120 <210> 177 <211> 5 <212> PRT <213> Homo sapiens <400> 177 Ser Tyr Tyr Ile His 1 5 <210> 178 <211> 17 <212> PRT <213> Homo sapiens <400> 178 Met Ile Asn Pro Ser Gly Gly Pro Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly      <210> 179 <211> 13 <212> PRT <213> Homo sapiens <400> 179 Glu Arg Trp Tyr Lys Trp Asn Phe Asp Ala Phe Asp Ile 1 5 10 <210> 180 <211> 30 <212> PRT <213> Homo sapiens <400> 180 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr             20 25 30 <210> 181 <211> 14 <212> PRT <213> Homo sapiens <400> 181 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 182 <211> 32 <212> PRT <213> Homo sapiens <400> 182 Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Asp 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Phe Tyr Cys Ala Arg             20 25 30 <210> 183 <211> 11 <212> PRT <213> Homo sapiens <400> 183 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 184 <211> 663 <212> DNA <213> Homo sapiens <400> 184 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccaac agaaaccagg acagcctcct aagctgctca tttactgggc atctacacgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc agcaggctga agatgtggca gtttattact gtcagcaata ttacagtact 300 cctcggacgt tcggccaagg gaccaaggtg gaaatcaaac gaactgtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaaga acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tag 663 <210> 185 <211> 339 <212> DNA <213> Homo sapiens <400> 185 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccaac agaaaccagg acagcctcct aagctgctca tttactgggc atctacacgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc agcaggctga agatgtggca gtttattact gtcagcaata ttacagtact 300 cctcggacgt tcggccaagg gaccaaggtg gaaatcaaa 339 <210> 186 <211> 51 <212> DNA <213> Homo sapiens <400> 186 aagtccagcc agagtgtttt atacagctcc aacaataaga actacttagc t 51 <210> 187 <211> 21 <212> DNA <213> Homo sapiens <400> 187 tgggcatcta cacgggaatc c 21 <210> 188 <211> 27 <212> DNA <213> Homo sapiens <400> 188 cagcaatatt acagtactcc tcggacg 27 <210> 189 <211> 69 <212> DNA <213> Homo sapiens <400> 189 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgc 69 <210> 190 <211> 45 <212> DNA <213> Homo sapiens <400> 190 tggtaccaac agaaaccagg acagcctcct aagctgctca tttac 45 <210> 191 <211> 96 <212> DNA <213> Homo sapiens <400> 191 ggggtccctg accgattcag tggcagcggg tctgggacag atttcactct caccatcagc 60 agccagcagg ctgaagatgt ggcagtttat tactgt 96 <210> 192 <211> 30 <212> DNA <213> Homo sapiens <400> 192 ttcggccaag ggaccaaggt ggaaatcaaa 30 <210> 193 <211> 220 <212> PRT <213> Homo sapiens <400> 193 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser             20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Gln Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 220 <210> 194 <211> 113 <212> PRT <213> Homo sapiens <400> 194 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser             20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln         35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val     50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Gln Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys      <210> 195 <211> 17 <212> PRT <213> Homo sapiens <400> 195 Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Ala      <210> 196 <211> 7 <212> PRT <213> Homo sapiens <400> 196 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 197 <211> 9 <212> PRT <213> Homo sapiens <400> 197 Gln Gln Tyr Tyr Ser Thr Pro Arg Thr 1 5 <210> 198 <211> 23 <212> PRT <213> Homo sapiens <400> 198 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys             20 <210> 199 <211> 15 <212> PRT <213> Homo sapiens <400> 199 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 200 <211> 32 <212> PRT <213> Homo sapiens <400> 200 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Gln Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys             20 25 30 <210> 201 <211> 10 <212> PRT <213> Homo sapiens <400> 201 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 202 <211> 13 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (1) <223> X = E or D <220> <221> MISC_FEATURE <222> (2) (2) X = Q, N, R, K or H <220> <221> MISC_FEATURE (222) (3) .. (3) X = A, G, W or Y <220> <221> MISC_FEATURE (222) (5) .. (5) X = W, Y, K, R or H <220> <221> MISC_FEATURE <222> (6) X = K, R, H, W, Y, T or S <220> <221> MISC_FEATURE (222) (7) .. (7) X = Y, W, N or Q <220> <221> MISC_FEATURE (222) (10) .. (10) X = A, G or V <400> 202 Xaa Xaa Xaa Tyr Xaa Xaa Xaa Phe Asp Xaa Phe Asp Ile 1 5 10 <210> 203 <211> 13 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE (222) (1) .. (1) <223> X = D or E <220> <221> MISC_FEATURE <222> (2) (2) X = Q or R <220> <221> MISC_FEATURE (222) (3) .. (3) X = A or W <220> <221> MISC_FEATURE (222) (5) .. (5) X = W or K <220> <221> MISC_FEATURE <222> (6) X = W or K <220> <221> MISC_FEATURE (222) (7) .. (7) X = Y or N <220> <221> MISC_FEATURE (222) (10) .. (10) X = A or V <400> 203 Xaa Xaa Xaa Tyr Xaa Xaa Xaa Phe Asp Xaa Phe Asp Ile 1 5 10

Claims (31)

An isolated human monoclonal antibody or antigen binding portion thereof that specifically binds human CCR2, wherein the antibody is
(a) binds an epitope comprised within the first extracellular loop of human CCR2;
(b) binds an epitope comprised within a second extracellular loop of human CCR2;
(c) An isolated human monoclonal antibody or antigen binding portion thereof, that binds to an epitope comprised in both the first extracellular loop and the second extracellular loop of human CCR2. The isolated human monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion possesses one or more of the following properties:
(a) has a selectivity for human CCR2 that is at least 100 times higher than its selectivity for human CCR5;
(b) binds to human CCR2 with a K _D of 2.0 × 10 ⁻⁷ M or less;
(c) competes with MCP-1 and / or MCP-3 for binding to CCR2;
characteristic of reducing the Ca ^{2 +} in response to a movement (d) MCP-1;
(e) reduce collagen I mRNA synthesis in response to MCP-1;
(f) reduce pERK phosphorylation in human monocytes in response to MCP-1;
(g) reduce the chemotaxis of human monocytes in response to MCP-1; And
(h) A characteristic of reducing actin polymerization in human monocytes in response to MCP-1. The method of claim 1, wherein the first extracellular loop of human CCR2 comprises the amino acid sequence of SEQ ID NO: 128; An isolated human monoclonal antibody or antigen binding portion thereof, wherein the second extracellular loop of human CCR2 comprises the amino acid sequence of SEQ ID NO: 129. (a) V _H CDR1 as set out in SEQ ID NO: 12, V _H CDR2 as set out in SEQ ID NO: 13 and V _H CDR3 as set out in SEQ ID NO: 14;
(b) V _H CDR1 as set out in SEQ ID NO: 48, V _H CDR2 as set out in SEQ ID NO: 49 and V _H CDR3 as set out in SEQ ID NO: 50;
(c) V _H CDR1 as set out in SEQ ID NO: 84, V _H CDR2 as set out in SEQ ID NO: 85 and V _H CDR3 as set out in SEQ ID NO: 86; And
(d) V _H CDR1 as set out in SEQ ID NO: 177, V _H CDR2 as set out in SEQ ID NO: 178 and V _H CDR3 as set out in SEQ ID NO: 179
An isolated human monoclonal antibody or antigen binding portion thereof that specifically binds human CCR2, comprising a heavy chain variable (V _H ) domain amino acid sequence comprising CDR1, CDR2 and CDR3 selected from the group consisting of: The method of claim 4, wherein
(a) V _L CDR1 as set out in SEQ ID NO: 30, V _L CDR2 as set out in SEQ ID NO: 31 and V _L CDR3 as set out in SEQ ID NO: 32;
(b) V _L CDR1 as set out in SEQ ID NO: 66, V _L CDR2 as set out in SEQ ID NO: 67 and V _L CDR3 as set out in SEQ ID NO: 68;
(c) V _L CDR1 as set out in SEQ ID NO: 102, V _L CDR2 as set out in SEQ ID NO: 103 and V _L CDR3 as set out in SEQ ID NO: 104; And
(d) V _L CDR1 as set out in SEQ ID NO: 195, V _L CDR2 as set out in SEQ ID NO: 196 and V _L CDR3 as set out in SEQ ID NO: 197
An isolated human monoclonal antibody or antigen binding portion thereof further comprising a light chain variable (V _L ) domain amino acid sequence comprising CDR1, CDR2 and CDR3 selected from the group consisting of: (a) V shown in SEQ ID NO: 30 _L CDR1, V _L CDR2 of SEQ ID NO: 31 and V _L of SEQ ID NO: 32 CDR3;
(b) V _L CDR1 as set out in SEQ ID NO: 66, V _L CDR2 as set out in SEQ ID NO: 67 and V _L CDR3 as set out in SEQ ID NO: 68;
(c) V _L CDR1 as set out in SEQ ID NO: 102, V _L CDR2 as set out in SEQ ID NO: 103 and V _L CDR3 as set out in SEQ ID NO: 104; And
(d) V _L CDR1 as set out in SEQ ID NO: 195, V _L CDR2 as set out in SEQ ID NO: 196 and V _L CDR3 as set out in SEQ ID NO: 197
An isolated human monoclonal antibody or antigen binding portion thereof that specifically binds human CCR2, comprising a light chain variable (V _L ) domain amino acid sequence comprising CDR1, CDR2 and CDR3 selected from the group consisting of: The V _H CDR1 of SEQ ID NO: 84, the V _H CDR2 of SEQ ID NO: 85, the V _H CDR3 of SEQ ID NO: 86, the V _L CDR1 of SEQ ID NO: 102, and the V _L CDR2 of SEQ ID NO: 103. And an isolated human monoclonal antibody or antigen binding portion thereof comprising V _L CDR3 set forth in SEQ ID NO: 104. An isolated human monoclonal antibody or antigen binding portion thereof that specifically binds human CCR2, comprising a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 202 and SEQ ID NO: 203. SEQ ID NO: 11, SEQ ID NO: 47, SEQ ID NO: 83, SEQ ID NO: 176, SEQ ID NO: 11 with conservative amino acid substitutions, SEQ ID NO: 47 with conservative amino acid substitutions, SEQ ID NO: 83 with conservative amino acid substitutions, conservative amino acid substitutions SEQ ID NO: 176 with SEQ ID NO: 162 compared to SEQ ID NO: 162; SEQ ID NO: 47 with germline amino acid substitutions compared to SEQ ID NO: 158; SEQ ID NO: 83 with germline amino acid substitutions compared to SEQ ID NO: 160 , SEQ ID NO: 176 having germline amino acid substitutions as compared to SEQ ID NO: 164, amino acid sequence at least 90% identical to SEQ ID NO: 11, amino acid sequence at least 90% identical to SEQ ID NO: 47, amino acid sequence at least 90% identical to SEQ ID NO: 83, Heavy chain variable (V _H ) domain selected from the group consisting of amino acid sequences at least 90% identical to the number 176 An isolated human monoclonal antibody or antigen binding portion thereof that specifically binds human CCR2, comprising an amino acid sequence that is a phosphorus. SEQ ID NO: 29, SEQ ID NO: 66, SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 194, SEQ ID NO: 29 with conservative amino acid substitutions, SEQ ID NO: 66 with conservative amino acid substitutions, SEQ ID NO: 101 with conservative amino acid substitutions, SEQ ID NO: 113 with conservative amino acid substitutions, SEQ ID NO: 194 with conservative amino acid substitutions, SEQ ID NO: 29 with germline amino acid substitutions compared to SEQ ID NO: 159, SEQ ID NO: 66 with germline amino acid substitutions compared to SEQ ID NO: 161, SEQ ID NO: 101 with germline amino acid substitutions in comparison to SEQ ID NO: 163, SEQ ID NO: 113 with germline amino acid substitutions in comparison to SEQ ID NO: 163, SEQ ID NO: 194 with germline amino acid substitutions in comparison to SEQ ID NO: 165, SEQ ID NOs: 29 and 90 Amino Acid Sequence More Than% Identical and 90% Equal To SEQ ID NO: 66 The same amino acid sequence, SEQ ID NO: 113 and at least 90% containing the same amino acid sequence and SEQ ID NO: 194 and light chain variable (V _L) domain amino acid sequence selected from the group consisting of the same amino acid sequence at least 90%, specifically the CCR2 enemy An isolated human monoclonal antibody or antigen binding portion thereof that binds. The method of claim 8, wherein SEQ ID NO: 29, SEQ ID NO: 66, SEQ ID NO: 101, SEQ ID NO: 113, SEQ ID NO: 194, SEQ ID NO: 29 with conservative amino acid substitutions, SEQ ID NO: 66 with conservative amino acid substitutions, conservative amino acid substitutions Germline amino acid substitutions as compared to SEQ ID NO: 101 with SEQ ID NO: 113 with conservative amino acid substitutions, SEQ ID NO: 194 with conservative amino acid substitutions, SEQ ID NO: 29 with germline amino acid substitutions; SEQ ID NO: 66 with germline amino acid substitutions in comparison to SEQ ID NO: 66, SEQ ID NO: 161, SEQ ID NO: 113 with germline amino acid substitutions in comparison with SEQ ID NO: 161, SEQ ID NO: 194 with germline amino acid substitutions in comparison with SEQ ID NO: 165 , An amino acid sequence of at least 90% identical to SEQ ID NO: 29, an amino acid sequence of at least 90% identical to SEQ ID NO: 66, No. 101 with the same amino acid sequence, SEQ ID NO: 113 with the same amino acid sequence and SEQ ID NO: 194 and 90% or more, further comprising a light chain variable (V _L) domain amino acid sequence selected from the group consisting of the same amino acid sequence at least 90% more than 90% Isolated human monoclonal antibody or antigen binding portion thereof. (a) independently selected from the heavy chain of an antibody selected from the group consisting of monoclonal antibody 4.40 (ATCC accession number PTA-6981), 4.22 (ATCC accession number PTA-6980), 4.39 and 4.9 (ATCC accession number PTA-6979) One or more heavy chain CDR1, CDR2 and / or CDR3; or
(b) one or more light chain CDR1, CDR2 and / or CDR3 independently selected from the light chain of the antibody selected from the group consisting of monoclonal antibodies 4.40, 4.22, 4.39 and 4.9; or
(c) one or more heavy chain CDR1, CDR2 and / or CDR3, and monoclonal antibodies 4.40, 4.22, 4.39 and 4.9 independently selected from the heavy chain of the antibody selected from the group consisting of monoclonal antibodies 4.40, 4.22, 4.39 and 4.9 At least one light chain CDR1, CDR2 and / or CDR3 independently selected from the light chain of an antibody selected from the group consisting of
An isolated human monoclonal antibody or antigen-binding portion thereof that specifically binds CCR2. SEQ ID NO: 10, SEQ ID NO: 46, SEQ ID NO: 82, SEQ ID NO: 116, SEQ ID NO: 175, SEQ ID NO: 10 with conservative amino acid substitutions, SEQ ID NO: 46 with conservative amino acid substitutions, SEQ ID NO: 82 with conservative amino acid substitutions, SEQ ID NO: 116 with conservative amino acid substitutions, SEQ ID NO: 175 with conservative amino acid substitutions, SEQ ID NO: 10 with germline amino acid substitutions compared to SEQ ID NO: 162, SEQ ID NO: 46 with germline amino acid substitutions compared to SEQ ID NO: 158, Selected from the group consisting of SEQ ID NO: 82 having germline amino acid substitutions compared to SEQ ID NO: 160, SEQ ID NO: 116 having germline amino acid substitutions compared to SEQ ID NO: 160, and SEQ ID NO: 175 having germline amino acid substitutions compared to SEQ ID NO: 164 Isolation that specifically binds to human CCR2, including a heavy chain having an amino acid sequence Human monoclonal antibodies. The method of claim 13, wherein SEQ ID NO: 28, SEQ ID NO: 64, SEQ ID NO: 100, SEQ ID NO: 112, SEQ ID NO: 193, SEQ ID NO: 28 with conservative amino acid substitutions, SEQ ID NO: 64 with conservative amino acid substitutions, conservative amino acid substitutions Germline amino acid substitutions compared to SEQ ID NO: 28 having SEQ ID NO: 100, conservative amino acid substitutions; SEQ ID NO: 193 with conservative amino acid substitutions, SEQ ID NO: 163; SEQ ID NO: 64 with SEQ ID NO: 64 with germline amino acid substitutions in comparison to SEQ ID NO: 161, SEQ ID NO: 112 with germline amino acid substitutions in comparison with SEQ ID NO: 161, and SEQ ID NO: 193 with germline amino acid substitutions in comparison with SEQ ID NO: 165 Isolated human further comprising a light chain having an amino acid sequence selected from the group consisting of One monoclonal antibody. The method of claim 14, wherein the antibody is
(a) a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 28,
(b) a heavy chain having the amino acid sequence of SEQ ID NO: 46 and a light chain having the amino acid sequence of SEQ ID NO: 64,
(c) a heavy chain having the amino acid sequence of SEQ ID NO: 82 and a light chain having the amino acid sequence of SEQ ID NO: 100,
(d) a heavy chain having the amino acid sequence of SEQ ID NO: 82 and a light chain having the amino acid sequence of SEQ ID NO: 112,
(e) a heavy chain having the amino acid sequence of SEQ ID NO: 116 and a light chain having the amino acid sequence of SEQ ID NO: 112; and
(f) a heavy chain having the amino acid sequence of SEQ ID NO: 175 and a light chain having the amino acid sequence of SEQ ID NO: 193
An isolated human monoclonal antibody or antigen binding portion thereof having a heavy and light chain selected from the group consisting of: The isolated human monoclonal antibody or antigen-binding portion thereof of claim 15, wherein the heavy chain amino acid sequence is SEQ ID NO: 116 and the light chain amino acid sequence is SEQ ID NO: 112. 16. The amino acid sequence of claim 5, wherein at least one V _H FR1, FR2, FR3 or FR4 and / or V _L FR1, FR2, FR3 or FR4 amino acid sequence of an antibody selected from the group consisting of 4.40, 4.40 A68G S230P, 4.39, 4.22 and 4.9. Isolated human monoclonal antibody or antigen binding portion thereof further comprising. 18. The isolated human monoclonal antibody or antigen binding portion thereof of claim 17, further comprising a heavy chain constant domain. 19. The isolated human monoclonal antibody or antigen binding portion thereof of claim 18, further comprising a light chain constant domain. A pharmaceutical composition comprising the antibody according to claim 1 or an antigen binding portion thereof and a pharmaceutically acceptable carrier. An isolated cell line producing the antibody according to claim 1 or an antigen binding portion thereof. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody according to claim 1 or an antigen binding portion thereof. A vector comprising the nucleic acid molecule according to claim 22 and optionally comprising an expression control sequence operably linked to the nucleic acid molecule. Treating, preventing, or treating the symptoms of a CCR2-mediated disease in a subject in need thereof, comprising administering an antibody or antigen-binding portion thereof according to claim 1 to a subject in need thereof. How to alleviate. The subject of claim 24, wherein the subject has an inflammatory disease, allergic disease, autoimmune disease, graft rejection, atherosclerosis, obesity, HIV infection, neuropathic pain, stenosis, restenosis, multiple sclerosis, fibrosis, aging And at least one condition or disease selected from the group consisting of macular degeneration, uveitis, corneal infection, and cancer. The method of claim 25, wherein the fibrosis is liver fibrosis. 27. The method of claim 26, wherein the liver fibrosis is due to hepatitis C virus (HCV) infection, hepatitis B virus (HBV) infection, alcoholic fatty hepatitis (ASH), or nonalcoholic fatty hepatitis (NASH). . Use of an antibody or antigen-binding portion thereof according to claim 1 for the manufacture of a medicament for treating, preventing or alleviating the symptoms of a CCR2-mediated disease in a subject in need thereof for treating, preventing or alleviating the symptoms of a CCR2-mediated disease. . The subject of claim 28, wherein the subject has an inflammatory disease, allergic disease, autoimmune disease, graft rejection, atherosclerosis, obesity, HIV infection, neuropathic pain, stenosis, restenosis, multiple sclerosis, fibrosis, aging Use of at least one condition or disease selected from the group consisting of macular degeneration, uveitis, corneal infection and cancer. 30. The use of claim 29, wherein the fibrosis is liver fibrosis. 31. The use of claim 30 wherein the liver fibrosis is due to hepatitis C virus (HCV) infection, hepatitis B virus (HBV) infection, alcoholic fatty hepatitis (ASH) or nonalcoholic fatty hepatitis (NASH). .

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